Anti-GAPDH antibody [6C5] - Loading Control

41 product images

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  Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Immunocytochemistry/ Immunofluorescence staining of SV40LT-SMC (Rat SV40-transfected aorta smooth cell line) cells using mouse Anti-GAPDH antibody

  ab8245 staining GAPDH in SV40LT-SMC (Rat SV40-transfected aorta smooth cell line) cells.

  The cells were fixed with 4% formaldehyde (10 minutes) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 5μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab202272 at 1/250 dilution overnight at +4°C followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) (shown in green). Nuclear DNA was labeled in blue with DAPI.

  Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

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  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  False colour image of Western blot: Anti-SIRPA antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to SIRPA. A band was observed at 100-140 kDa (mouse SIRPA, isoform 1), in wild-type RAW 264.7 cell lysates (band observed at 70-100 kDa in THP-1 is Human SIRPA) with no signal observed at this size in SIRPA knockout cell line Mouse SIRPA knockout RAW 264.7 cell line ab281618 (knockout cell lysate Mouse SIRPA knockout RAW 264.7 cell lysate ab282969). To generate this image, wild-type and SIRPA knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/20000 dilution

  Lane 1: Wild-type RAW 264.7 cell lysate at 20 µg

  Lane 2: RAW 264.7 cell lysate at 20 µg

  Lane 2: Western blot - Mouse SIRPA knockout RAW 264.7 cell line (Mouse SIRPA knockout RAW 264.7 cell line ab281618)

  Lane 3: THP-1 cell lysate at 20 µg

  Lane 4: MCF7 cell lysate at 20 µg

  Predicted band size: 55 kDa

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  This image is courtesy of an anonymous customer review

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  Lane 1: Mouse hippocampus whole cell lysate at 20 µg

  Lane 2: Rat hippocampus whole cell lysate at 20 µg

  Secondary

  All lanes: HRP-conjugated Rabbit anti-mouse at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 36 kDa

  Observed band size: 36 kDa

  Exposure time: 10s

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  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lanes 1-3: Merged signal (red and green). Green - Anti-nmt55 / p54nrb antibody [EPR5270] ab133574 observed at 63 kDa. Red - loading control, ab8245 observed at 37 kDa.

  Anti-nmt55 / p54nrb antibody [EPR5270] ab133574 Anti-nmt55 / p54nrb antibody [EPR5270] was shown to specifically react with nmt55 / p54nrb in wild-type HEK293T cells. Loss of signal was observed when knockout cell line Human NONO (nmt55 / p54nrb) knockout HEK-293T cell line ab266244 (knockout cell lysate Human NONO (nmt55 / p54nrb) knockout HEK-293T cell lysate ab257160) was used. Wild-type and nmt55 / p54nrb knockout samples were subjected to SDS-PAGE. Anti-nmt55 / p54nrb antibody [EPR5270] ab133574 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (Anti-nmt55 / p54nrb antibody [EPR5270] ab133574) at 1/1000 dilution

  Lane 1: Wild-type HEK293T cell lysate at 20 µg

  Lane 2: NONO knockout HEK293T cell lysate at 20 µg

  Lane 3: MOLT-4 cell lysate at 20 µg

  Secondary

  Lanes 1 - 3: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773)

  Lanes 1 - 3: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776)

  Performed under reducing conditions.

  Predicted band size: 54 kDa

  Observed band size: 63 kDa, 37 kDa

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  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  Lanes 1- 2: Merged signal (red and green). Green - Anti-Sortilin/NT3 antibody ab16640 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
  

  Anti-Sortilin/NT3 antibody ab16640 was shown to react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human SORT1 (Sortilin/NT3) knockout HeLa cell line ab264772 (knockout cell lysate Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) was used. Wild-type HeLa and SORT1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Sortilin/NT3 antibody ab16640 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Sortilin/NT3 antibody (Anti-Sortilin/NT3 antibody ab16640) at 1 µg/mL

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 92 kDa

  Observed band size: 100 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Immunocytochemistry/ Immunofluorescence staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells using mouse Anti-GAPDH antibody

  ab8245 staining GAPDH in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

  The cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 5 μg/ml and Anti-beta Tubulin antibody - Loading Control ab6046 at 1 μg/ml overnight at +4°C followed by a further incubation at room temperature for 1 hour with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

  Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

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  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HeLa cell lysate 20 μg
  Lane 2: NPR1 knockout HeLa cell lysate 20 μg
  Lane 3: U-251 MG cell lysate 20 μg
  Lane 4: Human Heart tissue lysate 20 μg
  

  
  Lanes 1 - 4: Merged signal (red and green). Green - anti-NPR1 antibody observed at 140 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
  anti-NPR1 antibody was shown to react with NPR1 in wild-type HeLa cells in Western blot with loss of signal observed in NPR1 knockout cell line Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell line ab265930 (NPR1 knockout cell lysate Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate ab258080). Wild-type HeLa and NPR1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with anti-NPR1 antibody and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4^°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  All lanes: anti-NPR1 antibody at 1/500 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: NPR1 knockout HeLa cell lysate at 20 µg

  Lane 3: U-251 MG cell lysate at 20 µg

  Lane 4: Human Heart tissue lysate at 20 µg

  Secondary

  Lanes 1 - 3: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 3: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

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  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  Lane 1: Wild-type HeLa cell lysate (20µg)
  
  Lane 2: SORT1 knockout HeLa cell lysate (20µg)
  
  Lanes 1- 2: Merged signal (red and green). Green - Anti-Sortilin/NT3 antibody [EPR15010] ab188586 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.
  
  Anti-Sortilin/NT3 antibody [EPR15010] ab188586 Anti-Sortilin/NT3 antibody [EPR15010] was shown to specifically react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human SORT1 (Sortilin/NT3) knockout HeLa cell line ab264772 (knockout cell lysate Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) was used. Wild-type and Sortilin/NT3 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-Sortilin/NT3 antibody [EPR15010] ab188586 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
  

  All lanes: Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (Anti-Sortilin/NT3 antibody [EPR15010] ab188586) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate ab257696) at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 92 kDa

  Observed band size: 100 kDa

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  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HeLa cell lysate (20µg)
  
  Lane 2: PMS2 knockout HeLa cell lysate (20µg)
  
  Lanes 1- 2: Merged signal (red and green). Green - Anti-PMS2 antibody [EPR3947] ab110638 observed at 120 kDa. Red - loading control ab8245 observed at 37 kDa.
  
  Anti-PMS2 antibody [EPR3947] ab110638 Anti-PMS2 antibody [EPR3947] was shown to specifically react with PMS2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human PMS2 knockout HeLa cell line ab261776 (knockout cell lysate Human PMS2 knockout HeLa cell lysate ab257142) was used. Wild-type and PMS2 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-PMS2 antibody [EPR3947] ab110638 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
  

  All lanes: Western blot - Anti-PMS2 antibody [EPR3947] (Anti-PMS2 antibody [EPR3947] ab110638) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: PMS2 knockout HeLa cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 96 kDa

  Observed band size: 120 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lanes 1-4: Merged signal (red and green). Green - Anti-MYL9 antibody [EPR13012(2)] ab191393 observed at 20 kDa. Red - loading control ab8245 observed at 37 kDa.

  Anti-MYL9 antibody [EPR13012(2)] ab191393 Anti-MYL9 antibody [EPR13012(2)] was shown to specifically react with MYL9 in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human MYL9 knockout HeLa cell line ab266036 (knockout cell lysate Human MYL9 knockout HeLa cell lysate ab256999) was used. Wild-type and MYL9 knockout samples were subjected to SDS-PAGE. Anti-MYL9 antibody [EPR13012(2)] ab191393 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-MYL9 antibody [EPR13012(2)] (Anti-MYL9 antibody [EPR13012(2)] ab191393) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: MYL9 knockout HeLa cell lysate at 20 µg

  Lane 3: Human colon cell lysate at 20 µg

  Lane 4: Daudi cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 20 kDa

  Observed band size: 20 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab140751 overnight at 4°C. Antibody binding was detected using the Donkey Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216779 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution

  Lane 1: Wild-type HAP1 cell lysate (20 µg)

  Lane 2: NF-κB p60 knockout HAP1 cell lysate (20 µg)

  Lane 3: HeLa cell lysate (20 µg)

  Lane 4: A431 cell lysate (20 µg)

  Secondary

  All lanes: Western blot - Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (Donkey Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216779) at 1/10000 dilution

  Predicted band size: 36 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with unpurified Anti-BDNF antibody [EPR1292] ab108319 (1/1000) overnight at 4°C. ab8245 (mouse anti-GAPDH; 0.05 ug/mL) was included as a loading control. Antibody binding was detected using goat anti-rabbit IgG IR-680 (green) and goat anti-mouse IgG IR800 (red) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx

  All lanes: Western blot - Anti-BDNF antibody [EPR1292] (Anti-BDNF antibody [EPR1292] ab108319) at 1/1000 dilution

  Lane 1: Human hippocampus lysate at 20 µg

  Lane 2: Rat hippocampus lysate at 20 µg

  Lane 3: Mouse hippocampus lysate at 20 µg

  Secondary

  All lanes: Gt anti Rb IR680 at 1/10000 dilution

  Performed under reducing conditions.

  Predicted band size: 27 kDa

  Observed band size: 15 kDa, 28 kDa, 35 kDa, 45 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HeLa cell lysate (20µg)
  
  Lane 2: IDH1 knockout HeLa cell lysate (20µg)
  
  Lanes 1- 2: Merged signal (red and green). Green - Anti-IDH1 antibody [EPR21002] ab230949 observed at 46 kDa. Red - loading control ab8245 observed at 37 kDa.
  
  Anti-IDH1 antibody [EPR21002] ab230949 Anti-IDH1 antibody [EPR21002] was shown to specifically react with IDH1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human IDH1 knockout HeLa cell line ab264916 (knockout cell lysate Human IDH1 knockout HeLa cell lysate ab257221) was used. Wild-type and IDH1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-IDH1 antibody [EPR21002] ab230949 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
  

  All lanes: Western blot - Anti-IDH1 antibody [EPR21002] (Anti-IDH1 antibody [EPR21002] ab230949) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: IDH1 knockout HeLa cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 46 kDa

  Observed band size: 46 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-Brd4 antibody [EPR5150(2)] ab128874 overnight at 4°C. Antibody binding was detected using the Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773. Loading control to GAPDH ab8245 antibody binding was detected using the Goat anti-Mouse IgG H&L (IRDye^® 680RD) (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
  

  Merged signal (red and green). Green Anti-Brd4 antibody [EPR5150(2)] Anti-Brd4 antibody [EPR5150(2)] ab128874 observed at 150 kDa using Goat anti-Rabbit IgG H&L (IRDye^® 800CW)-Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 as secondary antibody. Red - Anti-GAPDH antibody loading control, ab8245, observed at 37 kDa,

  Lane 1: Western blot - Anti-Brd4 antibody [EPR5150(2)] (Anti-Brd4 antibody [EPR5150(2)] ab128874)

  Lanes 1 - 3: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/10000 dilution

  Lane 1: Wild-type HAP1 cell lysate (20 µg)

  Lane 2: Brd4 knockout HAP1 cell lysate (20 µg)

  Lane 3: HeLa cell lysate (20 µg)

  Secondary

  All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution

  Predicted band size: 152 kDa, 36 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD13 antibody [SP187] ab227663 observed at 160 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
  

  Anti-CD13 antibody [SP187] ab227663 was shown to react with CD13 in wild-type THP-1 cells in western blot with loss of signal observed in ANPEP knockout cell line Human ANPEP (CD13) knockout THP-1 cell line ab273759 (knockout cell lysate Human ANPEP (CD13) knockout THP-1 cell lysate ab275505). Wild-type and ANPEP knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with Anti-CD13 antibody [SP187] ab227663 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 400 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-CD13 antibody [SP187] (Anti-CD13 antibody [SP187] ab227663) at 1/400 dilution

  Lane 1: Wild-type THP-1 cell lysate at 30 µg

  Lane 2: ANPEP knockout THP-1 cell lysate at 30 µg

  Lane 3: PANC-1 cell lysate at 30 µg

  Lane 4: HEK-293 cell lysate at 30 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 109 kDa

  Observed band size: 160 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HCT116 cell lysate 20 ug
  Lane 2: CTNNB1 knockout HCT116 cell lysate 20 ug
  Lanes 1 - 2: Merged signal (red and green). Green - Anti-beta Catenin antibody [IGX4794R-3] ab223075 observed at 95 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
  Anti-beta Catenin antibody [IGX4794R-3] ab223075 was shown to react with Anti-beta Catenin in wild-type HCT 116 cells in western blot with loss of signal observed in CTNNB1 knockout cell line Human CTNNB1 knockout HCT116 cell line ab273712 (CTNNB1 knockout cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). HCT 116 wild-type and CTNNB1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluoroscent western blot (TBS-based) blocking solution 50% (v/v) in TBS-T (0.1% Tween^®) before incubation with Anti-beta Catenin antibody [IGX4794R-3] ab223075 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4^°C at 1 ug/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-beta Catenin antibody [IGX4794R-3] (Anti-beta Catenin antibody [IGX4794R-3] ab223075) at 1 µg/mL

  Lane 1: Wild-type HCT116 cell lysate at 20 µg

  Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate (Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247)

  Lane 2: Western blot - Human CTNNB1 knockout HCT116 cell line (Human CTNNB1 knockout HCT116 cell line ab273712)

  Lane 2: CTNNB1 knockout HCT116 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 10 µg/mL

  All lanes: Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

  Predicted band size: 36 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lanes 1 - 4: Merged signal (red and green). Green - Anti-Cytokeratin 14 antibody [SP53] ab119695 observed at 52 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
  

  Anti-Cytokeratin 14 antibody [SP53] ab119695 was shown to react with KRT14 in A431 wild-type cells in Western blot. Loss of signal was observed when KRT14 knockout sample was used. A431 wild-type and KRT14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with Anti-Cytokeratin 14 antibody [SP53] ab119695 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 93 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Cytokeratin 14 antibody [SP53] (Anti-Cytokeratin 14 antibody [SP53] ab119695) at 1/93 dilution

  Lane 1: Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: KRT14 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 3: Human skin whole tissue lysate at 20 µg

  Lane 4: MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 52 kDa

  Observed band size: 52 kDa, 37 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Immunocytochemistry/ Immunofluorescence staining of NIH/3T3 (Mouse embryo fibroblast cell line) cells using mouse Anti-GAPDH antibody

  ab8245 staining GAPDH in NIH/3T3 (Mouse embryo fibroblast cell line) cells.

  The cells were fixed with 4% formaldehyde (10 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 1 μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab202272 at 1/250 dilution overnight at +4°C followed by a further incubation at room temperature for 1 hour with Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) (shown in green). Nuclear DNA was labeled in blue with DAPI.

  Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HCT 116 cell lysate 20 μg
  Lane 2: CTNNB1 knockout HCT 116 cell lysate 20 μg
  False colour image of Western blot: Anti-beta Catenin antibody [IGX4794R-3] staining at 1 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-beta Catenin antibody [IGX4794R-3] ab223075 was shown to bind specifically to beta Catenin. A band was observed at 95 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CTNNB1 knockout cell line Human CTNNB1 knockout HCT116 cell line ab273712 (knockout cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). The band observed in the knockout lysate lane below 95 kDa is likely to represent a truncated form of beta Catenin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CTNNB1 knockout HCT 116 cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-beta Catenin antibody [IGX4794R-3] (Anti-beta Catenin antibody [IGX4794R-3] ab223075) at 1 µg/mL

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate (Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247)

  Lane 2: Western blot - Human CTNNB1 knockout HCT116 cell line (Human CTNNB1 knockout HCT116 cell line ab273712)

  Lane 2: CTNNB1 knockout HCT 116 cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  Fluorescence detection of secondary antibody.

  All lanes: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 2.5 µg/mL

  Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) Nuclear at 20 µg

  Lane 2: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Lane 3: A431 (Human epidermoid carcinoma cell line) cell lysate at 20 µg

  Lane 4: Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 20 µg

  Lane 5: HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 20 µg

  Secondary

  All lanes: Alexa Fluor anti-mouse at 1/5000 dilution

  Performed under reducing conditions.

  Predicted band size: 36 kDa

  Observed band size: 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HeLa cell lysate (20 μg)
  

  Lane 2: EIF4EBP1 knockout HeLa cell lysate (20 μg)

  Lanes 1-2: Merged signal (red and green). Green - Anti-eIF4EBP1 antibody [Y329] ab32024 observed at 13 kDa. Red - loading control ab8245 observed at 37 kDa.

  Anti-eIF4EBP1 antibody [Y329] ab32024 Anti-eIF4EBP1 antibody [Y329] was shown to specifically react with eIF4EBP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human EIF4EBP1 knockout HeLa cell line ab264784 (knockout cell lysate Human EIF4EBP1 knockout HeLa cell lysate ab257146) was used. Wild-type and eIF4EBP1 knockout samples were subjected to SDS-PAGE. Anti-eIF4EBP1 antibody [Y329] ab32024 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 Dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-eIF4EBP1 antibody [Y329] (Anti-eIF4EBP1 antibody [Y329] ab32024) at 1/5000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: EIF4EBP1 knockout HeLa cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Lanes 1 - 3: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 13 kDa

  Observed band size: 13 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lanes 1- 2: Merged signal (red and green). Green - Anti-NFkB p100/NFKB2 antibody [EPR4686] ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
  

  Anti-NFkB p100/NFKB2 antibody [EPR4686] ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HepG2 cells in western blot. The band observed in CRISPR/Cas9 edited cell line Human NFKB2 (NFkB p100/NFKB2) knockout Hep G2 cell line ab262323 (CRISPR/Cas9 edited cell lysate Human NFKB2 (NFkB p100/NFKB2) knockout Hep G2 cell lysate ab257247) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HepG2 and NFKB2 CRISPR/Cas9 edited HepG2 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-NFkB p100/NFKB2 antibody [EPR4686] ab109440 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] (Anti-NFkB p100/NFKB2 antibody [EPR4686] ab109440) at 1/1000 dilution

  Lane 1: Wild-type HepG2 cell lysate at 20 µg

  Lane 2: NFKB2 CRISPR/Cas9 edited HepG2 cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 30 kDa, 97 kDa

  Observed band size: 120 kDa, 42 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  False colour image of Western blot: Anti-OXCT1/SCOT antibody staining at 0.04 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-OXCT1/SCOT antibody ab224250 was shown to bind specifically to OXCT1/SCOT. A band was observed at 56 kDa in wild-type HeLa cell lysates with no signal observed at this size in OXCT1 knockout cell line Human OXCT1 (SCOT) knockout HeLa cell line ab265358 (knockout cell lysate Human OXCT1 (SCOT) knockout HeLa cell lysate ab258557). To generate this image, wild-type and OXCT1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  Lanes 1 - 4: Western blot - Anti-OXCT1/SCOT antibody (Anti-OXCT1/SCOT antibody ab241125) at 1/10000 dilution

  Lanes 1 - 4: Western blot - Anti-OXCT1/SCOT antibody (Anti-OXCT1/SCOT antibody ab241221) at 1/10000 dilution

  Lanes 1 - 4: Western blot - Anti-OXCT1/SCOT antibody (Anti-OXCT1/SCOT antibody ab224250) at 0.04 µg/mL

  Lane 1: Wild-type HeLa cell lysate at 18 µg

  Lane 2: OXCT1 knockout HeLa cell lysate at 18 µg

  Lane 3: Jurkat cell lysate at 18 µg

  Lane 4: A549 cell lysate at 18 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 56 kDa

  Observed band size: 56 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Lane 1: Wild-type HeLa cell lysate 20 μg
  
  Lane 2: WWTR1 knockout HeLa cell lysate 20 μg
  
  Lane 3: A549 cell lysate 20 μg
  
  Lane 4: K562 cell lysate 20 μg
  
  False colour image of Western blot: Anti-TAZ antibody staining at 1 μg/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-TAZ antibody ab110239 was shown to bind specifically to TAZ. A band was observed at 52 kDa in wild-type HeLa cell lysates with no signal observed at this size in WWTR1 knockout cell line Human WWTR1 knockout HeLa cell line ab281598 (knockout cell lysate ab282950). To generate this image, wild-type and WWTR1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 ^°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-TAZ antibody (Anti-TAZ antibody ab110239) at 1 µg/mL

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: WWTR1 knockout HeLa cell lysate at 20 µg

  Lane 3: A549 cell lysate at 20 µg

  Lane 4: K562 cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 44 kDa

  Observed band size: 52 kDa, 37 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Anti-TRIM8 antibody [EPR27965-6] Anti-TRIM8 antibody [EPR27965-6] ab316149 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 65 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in TRIM8 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-TRIM8 antibody [EPR27965-6] (Anti-TRIM8 antibody [EPR27965-6] ab316149) at 1/1000 dilution

  Lane 1: Wild-type MCF7 at 20 µg

  Lane 2: TRIM8 knockout MCF7 at 20 µg

  Lane 3: OVCAR-3 at 20 µg

  Lane 4: HCT 116 at 20 µg

  Lane 5: HepG2 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 65 kDa

  Observed band size: 65 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: GLRX1 (E4D6J) Rabbit mAb ab92350S staining at 1/1000 dilution, shown in black; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 12 kDa in Wild-type A549 cell lysates with no signal observed at this size in GLRX knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit HRP (H+L) & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: GLRX knockout A549 at 20 µg

  Lane 3: RKO at 20 µg

  Lane 4: A549 Nuclear at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit HRP (H+L) & Goat anti-Mouse 680RD at 1/20000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Exposure time: 180s

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining of Wild-type A549 using mouse Anti-GAPDH antibody

  Western blot: DMT1/SLC11A2 (D3V8G) Rabbit mAb ab15083S staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 60-90 kDa in Wild-type A549 cell lysates with no signal observed at this size in SLC11A2 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: SLC11A2 knockout A549 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Monoclonal[E343] to S6K1 Anti-S6K1 antibody [E343] ab32529 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 59 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in RPS6KB1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-S6K1 antibody [E343] (Anti-S6K1 antibody [E343] ab32529) at 1/10000 dilution

  Lane 1: Wild-type U-87 MG at 20 µg

  Lane 2: RPS6KB1 knockout U-87 MG at 20 µg

  Lane 3: MCF7 at 20 µg

  Lane 4: HEK-293 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 59 kDa

  Observed band size: 59 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Monoclonal[EPR27133-30] to RNF5/NG2 Anti-RNF5/NG2 antibody [EPR27133-30] ab308066 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 18 kDa in Wild-type A549 cell lysates with no signal observed at this size in RNF5 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-RNF5/NG2 antibody [EPR27133-30] (Anti-RNF5/NG2 antibody [EPR27133-30] ab308066) at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: RNF5 knockout A549 at 20 µg

  Lane 3: SH-SY5Y at 20 µg

  Lane 4: A-498 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 20 kDa

  Observed band size: 18 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Recombinant Anti-SREBP1 antibody [EPR27079-194] Anti-SREBP1 antibody [EPR27079-194] ab313881 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 125 kDa in Wild-type HeLa cell lysates with no signal observed at this size in SREBF1 knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-SREBP1 antibody [EPR27079-194] (Anti-SREBP1 antibody [EPR27079-194] ab313881) at 1/1000 dilution

  Lane 1: Wild-type HeLa at 20 µg

  Lane 2: Western blot - Human SREBF1 (SREBP1) knockout HeLa cell line (Human SREBF1 (SREBP1) knockout HeLa cell line ab277151) at 20 µg

  Lane 3: Wild-type HCT 116 ab288559 at 20 µg

  Lane 4: Western blot - Human SREBF1 knockout HCT116 cell line (Human SREBF1 knockout HCT116 cell line ab289113) at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 87 kDa

  Observed band size: 87 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  All lanes: Western blot - Anti-KIF5A antibody (Anti-KIF5A antibody ab5628) at 1/1000 dilution

  Lane 1: Wild-type U-87 MG ab278079 at 20 µg

  Lane 2: Western blot - Human KIF5A knockout U-87 MG cell line (Human KIF5A knockout U-87 MG cell line ab306717) at 20 µg

  Lane 3: SK-N-FI at 20 µg

  Lane 4: HEK-293T at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 117 kDa

  Observed band size: 135 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Monoclonal[EP1255Y] to MMP9 Anti-MMP9 antibody [EP1255Y] ab137867 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 78 kDa in Wild-type A549 TPA-treated (80nM, 24h) cell lysates with no signal observed at this size in Wild-type A549 Vehicle Control TPA (0nM, 24h) cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-MMP9 antibody [EP1255Y] (Anti-MMP9 antibody [EP1255Y] ab137867) at 1/1000 dilution

  Lane 1: Wild-type A549 TPA-treated (80nM, 24h) at 20 µg

  Lane 2: Wild-type A549 Vehicle Control TPA (0nM, 24h) at 20 µg

  Lanes 3 - 4: Western blot - Human MMP9 knockout A549 cell line (ab286527) at 20 µg

  Lane 5: Human Lung at 20 µg

  Lane 6: MOLT-4 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 78 kDa

  Observed band size: 78 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Polyclonal to TTC11/FIS1 229969 staining at 1/2000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 16 kDa in Wild-type Jurkat cell lysates with no signal observed at this size in FIS1 knockout Jurkat cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-TTC11/FIS1 antibody (Anti-TTC11/FIS1 antibody ab229969) at 1/2000 dilution

  Lane 1: Wild-type Jurkat at 20 µg

  Lane 2: FIS1 knockout Jurkat at 20 µg

  Lane 3: HeLa at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 17 kDa

  Observed band size: 16 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  False colour image of Western blot: Anti-Nrf2 antibody [EP1808Y] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-Nrf2 antibody [EP1808Y] ab62352 was shown to bind specifically to Nrf2.
  

  Target band (indicated by arrow) was observed at 85-90 kDa in wild-type A549 cell lysates with no signal observed at this size in NFE2L2 knockout cell line Human NFE2L2 knockout A549 cell line[21] ab285359 (knockout cell lysate ab289682). A band lower than the target was present on both WT and KO. According to the literature (PMID: 40043449), the lower band is calmegin.

  To generate this image, wild-type and NFE2L2 knockout A549 cell lysates were analysed. Please note that MG132 treatment does not affect expression levels of Nrf2. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-Nrf2 antibody [EP1808Y] (Anti-Nrf2 antibody [EP1808Y] ab62352) at 1/1000 dilution

  Lane 1: Wild-type A549 Vehicle control MG132 (0 uM, 18 h) cell lysate at 20 µg

  Lane 2: Wild-type A549 Treated MG132 (2 uM, 18 h) cell lysate at 20 µg

  Lane 3: NFE2L2 [21] knockout A549 Vehicle control MG132 (0 uM, 18 h) cell lysate at 20 µg

  Lane 4: NFE2L2 [21] knockout A549 Treated MG132 (2 uM, 18 h) cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 68 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Polyclonal to Furin Anti-Furin antibody ab3467 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 87 kDa in Wild-type A549 cell lysates with no signal observed at this size in FURIN knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-Furin antibody (Anti-Furin antibody ab3467) at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: FURIN knockout A549 at 20 µg

  Lane 3: HEK-293 at 20 µg

  Lane 4: MOLT-4 at 20 µg

  Lane 5: Ramos at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 87 kDa

  Observed band size: 87 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: anti-Furin antibody staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 87 kDa in Wild-type A549 cell lysates with no signal observed at this size in FURIN knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot at 1/1000 dilution

  Lane 1: Wild-type A549 at 20 µg

  Lane 2: FURIN knockout A549 at 20 µg

  Lane 3: HEK-293 at 20 µg

  Lane 4: MOLT-4 at 20 µg

  Lane 5: Ramos at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 87 kDa

  Observed band size: 87 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  All lanes: Western blot - Anti-KIF5A antibody (Anti-KIF5A antibody ab154378) at 1/1000 dilution

  Lane 1: Wild-type U-87 MG ab278079 at 20 µg

  Lane 2: Western blot - Human KIF5A knockout U-87 MG cell line (Human KIF5A knockout U-87 MG cell line ab306717) at 20 µg

  Lane 3: SK-N-FI at 20 µg

  Lane 4: HEK-293T at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 117 kDa

  Observed band size: 135 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  All lanes: Western blot - Anti-MeCP2 antibody - ChIP Grade (Anti-MeCP2 antibody - ChIP Grade ab195393) at 1/1000 dilution

  Lane 1: Wild-type HCT 116 ab288559 at 20 µg

  Lane 2: Western blot - Human MECP2 knockout HCT116 cell line (Human MECP2 knockout HCT116 cell line ab289193) at 20 µg

  Lane 3: Wild-type HAP1 at 20 µg

  Lane 4: MECP2 knockout HAP1 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 52 kDa

  Observed band size: 52 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Monoclonal[E175] to S6K1 Anti-S6K1 antibody [E175] ab32359 staining at 1/5000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 60 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in RPS6KB1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-S6K1 antibody [E175] (Anti-S6K1 antibody [E175] ab32359) at 1/5000 dilution

  Lane 1: Wild-type U-87 MG at 20 µg

  Lane 2: RPS6KB1 knockout U-87 MG at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 59 kDa

  Observed band size: 60 kDa

• 

  Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)

  GAPDH Western blot staining using mouse Anti-GAPDH antibody

  Western blot: Rabbit Monoclonal[EPR4130(3)] to PICK1 Anti-PICK1 antibody [EPR4130(3)] ab133773 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 47 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in PICK1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

  All lanes: Western blot - Anti-PICK1 antibody [EPR4130(3)] (Anti-PICK1 antibody [EPR4130(3)] ab133773) at 1/1000 dilution

  Lane 1: Wild-type U-87 MG at 20 µg

  Lane 2: PICK1 knockout U-87 MG at 20 µg

  Lane 3: T-47D at 20 µg

  Lane 4: MCF7 at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 47 kDa

  Observed band size: 47 kDa