Anti-Vinculin antibody [EPR8185] - Loading Control

41 product images

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] (ab129002)

  Immunofluorescent staining of HEK293 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab129002 at a dilution of 1/50. An Alexa Fluor^® 488 goat anti-rabbit antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary at a dilution of 1/1000 and the cells were counter stained with DAPI. The negative controls are shown in the bottom middle and right hand panels. For negative control 1, the primary was used and then goat anti-mouse IgG was used at a dilution of 1/500. For negative control 2, a mouse primary antibody (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) and anti-rabbit secondary antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) were used.

• 

  Immunoprecipitation - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  ab129002 (purified) at 1/20 immunoprecipitating vinculin in HeLa cells. Lane 1: HeLa whole cell lysate (10 μg). Lane 2: HeLa whole cell lysate (10 μg). Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab129002 in HeLa whole cell lysate. For western blotting, a HRP-conjugated goat anti-rabbit antibody was used as the secondary antibody (1/1000).

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Predicted band size: 124 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking buffer: 5% NFDM/TBST

  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) at 1/10000 dilution

  Lane 1: HepG2 cell lysate at 20 µg

  Lane 2: HeLa cell lysate at 20 µg

  Lane 3: PC3 cell lysate at 20 µg

  Secondary

  All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

  Predicted band size: 124 kDa

  Observed band size: 124 kDa

• 

  Flow Cytometry (Intracellular) - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Flow Cytometry (Intracellular) staining using rabbit Anti-Vinculin antibody

  Intracellular Flow Cytometry analysis of 293 (human embryonic kidney epithelial) cells labeling Vinculin (red) with ab129002 at a 1/200 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.
  

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Different batches of ab129002 were tested on HepG2 (Human hepatocellular carcinoma epithelial cell) lysate at 1.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 124 kDa.

  All lanes: Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Predicted band size: 124 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking buffer: 5% NFDM/TBST

  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) at 1/20000 dilution

  Lane 1: C6 cell lysate at 10 µg

  Lane 2: rat kidney cell lysate at 10 µg

  Secondary

  All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

  Predicted band size: 124 kDa

  Observed band size: 124 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-Vinculin antibody

  Immunofluorescent staining of vinculin in HUVEC cells with unpurified ab129002 at 1/100 dilution.

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking buffer: 5% NFDM/TBST

  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) at 1/10000 dilution

  Lane 1: mouse spleen at 10 µg

  Lane 2: NIH/3T3 cell lysate at 10 µg

  Secondary

  All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

  Predicted band size: 124 kDa

  Observed band size: 124 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  All lanes: Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) at 1/10000 dilution

  Lane 1: PC3 cell lysate at 10 µg

  Lane 2: HeLa cell lysate at 10 µg

  Lane 3: U937 cell lysate at 10 µg

  Lane 4: K562 cell lysate at 10 µg

  Lane 5: HUVEC cell lysate at 10 µg

  Lane 6: Human fetal liver lysate at 10 µg

  Lane 7: Human fetal kidney lysate at 10 µg

  Secondary

  All lanes: Goat anti-Rabbit HRP at 1/2000 dilution

  Predicted band size: 124 kDa

  Observed band size: 124 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: NIH/3T3.
  
  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 34667079).
  
  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-TET2 antibody [EPR26694-93] (Anti-TET2 antibody [EPR26694-93] ab309481) at 1/1000 dilution

  Lane 1: F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 260 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 6 seconds

  All lanes: Western blot - Anti-BCAT2 antibody [EPR27488-91] (Anti-BCAT2 antibody [EPR27488-91] ab309514) at 1/1000 dilution

  Lane 1: Human skeletal muscle tissue lysate at 20 µg

  Lane 2: Human liver tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 37 kDa

  Exposure time: 6s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  Negative control: spleen (PMID: 24309898; PMID:24386422).

  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10, 000 dilution.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  All lanes: Western blot - Anti-Tau (MBD region) antibody [EPR25205-233] (Anti-Tau (MBD region) antibody [EPR25205-233] ab308439) at 1/1000 dilution

  Lane 1: Mouse brain tissue lysate at 20 µg

  Lane 2: Mouse spleen tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Developed using the ECL technique.

  Predicted band size: 78 kDa

  Observed band size: 50 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Low expression: liver (PMID: 11673838), kidney (PMID: 1673838).
  
  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11021599).
  
  The bands beneath the target band (320 kDa) are likely to be degraded target fragments.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-Collagen VII antibody [EPR27012-157] (Anti-Collagen VII antibody [EPR27012-157] ab309143) at 1/1000 dilution

  Lane 1: Mouse skin tissue lysate at 20 µg

  Lane 2: Mouse liver tissue lysate at 20 µg

  Lane 3: Mouse kidney tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 320 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Low expression: HCT116, HEK-293 (PMID: 23743647).
  
  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11021599).
  
  
  In lane 1, the lysate was stored at -80? prior to Western Blotting. The bands beneath the target band (320 kDa) are likey to be degradation products. In lanes 2-4, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-Collagen VII antibody [EPR27012-157] (Anti-Collagen VII antibody [EPR27012-157] ab309143) at 1/1000 dilution

  Lane 1: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 40 µg

  Lane 2: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 3: HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg

  Lane 4: HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 320 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Performed under reducing conditions.
  In Western blot, Anti-PHIP antibody [EPR27150-82] ab309962 was shown to bind specifically to PHIP. A band was observed at 250 kDa in wild-type Hela cell lysates whereas no signal observed at this size in PHIP knockout cell line Human PHIP knockout HeLa cell line ab265403 (knockout cell lysate Human PHIP knockout HeLa cell lysate ab259039).
  The band beneath the target band (250 kDa) is likely to be degraded target fragment.
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 3 minutes

  All lanes: Western blot - Anti-PHIP antibody [EPR27150-82] (Anti-PHIP antibody [EPR27150-82] ab309962) at 1/1000 dilution

  Lane 1: Wild-type HeLa(human cervical adenocarcinoma epithelial cell) whole cell lysate at 40 µg

  Lane 2: Western blot - Human PHIP knockout HeLa cell lysate (Human PHIP knockout HeLa cell lysate ab259039) at 40 µg

  Lane 3: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 250 kDa

  Exposure time: 3min

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Negative control: spleen
  
  The blot developed using a high sensitivity ECL substrate.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-GLS2 antibody [EPR27332-67] (Anti-GLS2 antibody [EPR27332-67] ab308304) at 1/1000 dilution

  Lane 1: Mouse pancreas tissue lysate at 20 µg

  Lane 2: Mouse spleen tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 66 kDa

  Observed band size: 66 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Low expression tissues: Kidney and heart (PMID:11604140).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  
  Exposure time: 1 second.

  All lanes: Western blot - Anti-Synaptophysin antibody [YE269] - Mouse IgG1 (Chimeric) (Anti-Synaptophysin antibody [YE269] - Mouse IgG1 (Chimeric) ab309493) at 1/1000 dilution

  Lane 1: Mouse brain tissue lysate at 20 µg

  Lane 2: Mouse cerebellum tissue lysate at 20 µg

  Lane 3: Mouse hippocampus tissue lysate at 20 µg

  Lane 4: Mouse heart tissue lysate at 20 µg

  Lane 5: Mouse kidney tissue lysate at 20 µg

  Lane 6: Rat brain tissue lysate at 20 µg

  Lane 7: Rat cerebellum tissue lysate at 20 µg

  Lane 8: Rat heart tissue lysate at 20 µg

  Lane 9: Rat kidney tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (Goat Anti-Mouse IgG Fc (HRP) preadsorbed ab98717) at 1/20000 dilution

  Observed band size: 38 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  The bands beneath the target band (400 kDa) are likely to be degraded target fragments.
  
  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: Lane1:180 seconds, lane2:15 seconds

  All lanes: Western blot - Anti-Plectin antibody [EPR26993-49] (Anti-Plectin antibody [EPR26993-49] ab312312) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

  Lane 2: U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 400-500 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Negative control: Saos-2 (PMID: 17202229; PMID: 9786925).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  
  Exposure time: 180 seconds.

  All lanes: Western blot - Anti-p53 antibody [G59-12] (Anti-p53 antibody [G59-12] ab308609) at 1/1000 dilution

  Lane 1: HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

  Lane 2: Saos-2 (human osteosarcoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

  Observed band size: 53 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST. Negative control: lung, liver (PMID: 9446595).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10, 000 dilution.
  
  Exposure time: 3 seconds

  All lanes: Western blot - Anti-Synaptogyrin 1 antibody [EPR25081-18] (Anti-Synaptogyrin 1 antibody [EPR25081-18] ab308617) at 1/1000 dilution

  Lane 1: Human hypothalamus tissue lysate at 20 µg

  Lane 2: Human lung tissue lysate at 20 µg

  Lane 3: Human liver tissue lysate at 20 µg

  Lane 4: Mouse brain tissue lysate at 20 µg

  Lane 5: Mouse hypothalamus tissue lysate at 20 µg

  Lane 6: Mouse lung tissue lysate at 20 µg

  Lane 7: Rat brain tissue lysate at 20 µg

  Lane 8: Rat lung tissue lysate at 20 µg

  Lane 9: Rat hypothalamus tissue lysate at 20 µg

  Secondary

  Lanes 1 - 9: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/2000 dilution

  Lanes 1 - 9: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/20000 dilution

  Predicted band size: 25 kDa

  Observed band size: 26 kDa

  Exposure time: 3s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 9837916, 10225972).

  Low expression: lung.

  The identity of the lower MW band at approximately 150 kDa is unknown.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.

  All lanes: Western blot - Anti-Apolipoprotein B antibody [EPR27473-49] (Anti-Apolipoprotein B antibody [EPR27473-49] ab312318) at 1/1000 dilution

  Lane 1: Human serum lysate at 20 µg

  Lane 2: Human plasma lysate at 20 µg

  Lane 3: Human lu lysate at 20 µg

  Lane 4: Mouse serum lysate at 20 µg

  Lane 5: Mouse plasma lysate at 20 µg

  Lane 6: Mouse lu lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Developed using the ECL technique.

  Observed band size: 250, 509 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 9837916, 10225972).

  Negative control: Hela (PMID: 2501159).

  The identity of the lower MW bands at approximately 300 kDa and 150 kDa is unknown.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.

  All lanes: Western blot - Anti-Apolipoprotein B antibody [EPR27473-49] (Anti-Apolipoprotein B antibody [EPR27473-49] ab312318) at 1/1000 dilution

  Lane 1: HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: Hela (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Observed band size: 509 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: K-562 (PMID: 14701728).
  
  The molecular weight observed is consistent with what has been described in the literature (PMID: 33692421; 8390468).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 103 seconds.

  All lanes: Western blot - Anti-BASP1 antibody [EPR28065-75] (Anti-BASP1 antibody [EPR28065-75] ab312322) at 1/1000 dilution

  Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

  Lane 2: HeLa transfected with siRNA 1 specifically targeti BASP1 whole cell lysate at 20 µg

  Lane 3: HeLa transfected with siRNA 2 specifically targeti BASP1 whole cell lysate at 20 µg

  Lane 4: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 55 kDa

  Exposure time: 103s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 8 seconds.

  All lanes: Western blot - Anti-ACAT1 antibody [EPR28035-8] (Anti-ACAT1 antibody [EPR28035-8] ab312320) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

  Lane 2: NIH/3T3 transfected with siRNA specifically targeti ACAT1 whole cell lysate at 20 µg

  Lane 3: Mouse liver tissue lysate at 20 µg

  Lane 4: Rat liver tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 45 kDa

  Exposure time: 8s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Anti-TOMM20 antibody [EPR15581-54] (Anti-TOMM20 antibody [EPR15581-54] - Mitochondrial Marker ab186735).
  
  In Western blot, anti-TOMM20 antibody [EPR15581-54] (Anti-TOMM20 antibody [EPR15581-54] - Mitochondrial Marker ab186735) staining at 1/1000 dilution. Anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 10 seconds.

  All lanes: Western blot - Anti-ACAT1 antibody [EPR28035-8] (Anti-ACAT1 antibody [EPR28035-8] ab312320) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast) non-mitochondrial fraction at 20 µg

  Lane 2: NIH/3T3 mitochondria fraction at 20 µg

  Lane 3: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 4: HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 5: A549 (human lu carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 6: PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 45 kDa

  Exposure time: 10s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative controls: brain (PMID: 24309898), kidney (PMID: 24309898), cerebellum (PMID: 24309898).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 48 seconds

  All lanes: Western blot - Anti-Neutrophil Elastase antibody [EPR28386-66] (Anti-Neutrophil Elastase antibody [EPR28386-66] ab310335) at 1/1000 dilution

  Lane 1: Mouse bone marrow tissue lysate at 20 µg

  Lane 2: Mouse cerebellum tissue lysate at 20 µg

  Lane 3: Mouse kidney tissue lysate at 20 µg

  Lane 4: Mouse brain tissue lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 29 kDa

  Exposure time: 48s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST,
  
  ab129002 was used as a loading control for Vinculin.

  All lanes: Western blot - Anti-Galectin 3 antibody [EP2775Y] (Anti-Galectin 3 antibody [EP2775Y] ab76245) at 1/1000 dilution

  Lane 1: RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

  Lane 2: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 3: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 5: SK-N-MC (human brain epithelial cell) whole cell lysate at 20 µg

  Lane 6: LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 26 kDa

  Observed band size: 26 kDa

  Exposure time: 26s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  In Western blot, anti-His antibody (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.
  Exposure time: 6 seconds

  All lanes: Western blot - Anti-SYT13 antibody [EPR26923-67] (Anti-SYT13 antibody [EPR26923-67] ab313329) at 1/1000 dilution

  Lane 1: 293T cells transfected with an empty vector containi a His-tag whole cell lysate at 20 µg

  Lane 2: 293T cells transfected with a Human SYT13 expression vector containi a His-tag whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 55 kDa

  Exposure time: 6s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Performed under reducing conditions.
  In Western blot, Anti-DR4 antibody [EPR28152-57] ab312848 was shown to bind specifically to TNFRSF10A (DR4). A band was observed at 45 kDa in wild-type Hela cell lysates whereas loss of signal was observed in the TNFRSF10A (DR4) knockout cell line Human TNFRSF10A (DR4) knockout HeLa cell line ab264687 (knockout cell lysate Human TNFRSF10A (DR4) knockout HeLa cell lysate ab258726).
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 15 seconds

  All lanes: Western blot - Anti-DR4 antibody [EPR28152-57] (Anti-DR4 antibody [EPR28152-57] ab312848) at 1/1000 dilution

  Lane 1: Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

  Lane 2: Western blot - Human TNFRSF10A (DR4) knockout HeLa cell lysate (Human TNFRSF10A (DR4) knockout HeLa cell lysate ab258726) at 50 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 45 kDa

  Exposure time: 15s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Low expression: kidney (PMID 33408860).
  This blot of Lane 6 was developed using a high sensitivity ECL substrate.
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: Lane 1-5: 180 seconds; Lane 6: 114 seconds.

  All lanes: Western blot - Anti-Sialoadhesin/CD169 antibody [EPR27102-11] (Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840) at 1/1000 dilution

  Lane 1: Mouse spleen tissue lysate at 20 µg

  Lane 2: Mouse lymph node tissue lysate at 20 µg

  Lane 3: Mouse kidney tissue lysate at 20 µg

  Lane 4: Rat lymph node tissue lysate at 20 µg

  Lane 5: Rat kidney tissue lysate at 20 µg

  Lane 6: Rat spleen tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 250 kDa

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  The expression of Sialoadhesin/CD169 is upregulated in response to LPS treatment (PMID: 27129263).
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-Sialoadhesin/CD169 antibody [EPR27102-11] (Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840) at 1/1000 dilution

  Lane 1: Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

  Lane 2: RAW 264.7 treated with /ml LPS for 16h whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 250 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  The bands beneath the target band (400 kDa) are likely to be degraded target fragments.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-Plectin antibody [EPR26993-49] (Anti-Plectin antibody [EPR26993-49] ab312312) at 1/1000 dilution

  Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

  Lane 2: HeLa transfected with siRNA specifically targeti plectin whole cell lysate 20 at 20 µg

  Secondary

  All lanes: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 400-500 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: skeletal muscle.
  Samples are non-boiled as boiling may cause protein aggregation.
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 3 seconds

  All lanes: Western blot - Anti-CLEC5A antibody [EPR28104-85] (Anti-CLEC5A antibody [EPR28104-85] ab313337) at 1/1000 dilution

  Lane 1: Mouse bone marrow tissue lysate at 20 µg

  Lane 2: Mouse skeletal muscle tissue lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 37 kDa

  Exposure time: 3s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: skeletal muscle.
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 37 seconds

  All lanes: Western blot - Anti-Reelin antibody [EPR26278-30] (Anti-Reelin antibody [EPR26278-30] ab312310) at 1/1000 dilution

  Lane 1: Rat cerebellum tissue lysate at 20 µg

  Lane 2: Rat hippocampus tissue lysate at 20 µg

  Lane 3: Rat skeletal muscle tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 420 kDa

  Exposure time: 37s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: skeletal muscle tissue (PMID: 22081212), liver (PMID: 10842222).
  
  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 16912065)(PMID: 35055012).
  
  SCN3A is abundantly expressed in neuronal tissues during embryonic and neonatal stages of development and is rare in adult tissues. (PMID: 29466837, 2555170).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-SCN3A antibody [EPR25135-66] (Anti-SCN3A antibody [EPR25135-66] ab309473) at 1/1000 dilution

  Lane 1: Rat E15 brain tissue lysate at 50 µg

  Lane 2: Rat skeletal muscle tissue lysate at 50 µg

  Lane 3: Rat liver tissue lysate at 50 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 260 kDa, 320 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: PANC-1 (PMID: 12531476), MCF7 (PMID: 28152297), SK-BR-3 (PMID: 28152297).
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-DDX53/CT26 antibody [EPR27384-107] (Anti-DDX53/CT26 antibody [EPR27384-107] ab313333) at 1/1000 dilution

  Lane 1: K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 50 µg

  Lane 2: PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 50 µg

  Lane 3: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 50 µg

  Lane 4: SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate at 50 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 74 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  Performed under reducing conditions.

  In Western blot, Anti-CD73 antibody [EPR28213-52] ab313339 was shown to bind specifically to CD73. A band was observed at 68 kDa in wild-type A431 cell lysates whereas loss of signal was observed in the CD73 knockout cell line.

  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  All lanes: Western blot - Anti-CD73 antibody [EPR28213-52] (Anti-CD73 antibody [EPR28213-52] ab313339) at 1/1000 dilution

  Lane 1: Wild-type A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 40 µg

  Lane 2: CD73 knockout A431 whole cell lysate at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 63 kDa

  Observed band size: 68 kDa

  Exposure time: 92s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-FIP200 antibody [EPR26076-87] (Anti-FIP200 antibody [EPR26076-87] ab313620) at 1/1000 dilution

  Lane 1: Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 50 µg

  Lane 2: HeLa treated with 2uM bafilomycin A1 for 18h whole cell lysate at 50 µg

  Lane 3: Untreated MEF (mouse embryo fibroblast) whole cell lysate at 50 µg

  Lane 4: MEF treated with 2uM bafilomycin A1 for 18h whole cell lysate at 50 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 200 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  The identity of the lower MW bands at approximately 100 kDa are unknown.

  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.

  All lanes: Western blot - Anti-FIP200 antibody [EPR26076-87] (Anti-FIP200 antibody [EPR26076-87] ab313620) at 1/1000 dilution

  Lane 1: Untreated A-172 (human brain glioblastoma ) whole cell lysate at 50 µg

  Lane 2: A-172 treated with 50uM chloroquine for 8h whole cell lysate at 50 µg

  Lane 3: Untreated C2C12 (mouse myoblast) whole cell lysate at 50 µg

  Lane 4: C2C12 treated with 50uM chloroquine for 24h whole cell lysate at 50 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Observed band size: 200 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Negative control: liver, lung, spleen (PMID: 7573490).
  
  The expression profile/ molecular weight observed is consistent with what has been described in literature (PMID: 22759959).
  
  In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.
  
  Samples are non-boiled as boiling may cause protein aggregation.
  Exposure time: 180 seconds

  All lanes: Western blot - Anti-SLC12A1/NKCC2 antibody [EPR28176-90] (Anti-SLC12A1/NKCC2 antibody [EPR28176-90] ab313640) at 1/1000 dilution

  Lane 1: Mouse kidney tissue lysate at 20 µg

  Lane 2: Mouse liver tissue lysate at 20 µg

  Lane 3: Mouse spleen tissue lysate at 20 µg

  Lane 4: Mouse lu tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 150, 270 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002)

  Vinculin Western blot staining using rabbit Anti-Vinculin antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Exposure time: Lane 1-2: 180 seconds; Lane 3-4: 81 seconds.
  
  Negative control: mouse liver and rat liver.
  In Western blot, anti-Vinculin antibody (ab129002) staining at 1/10, 000 dilution.
  Samples are non-boiled as boiling may cause protein aggregation.

  All lanes: Western blot - Anti-Dystrophin antibody [EPR21189] (Anti-Dystrophin antibody [EPR21189] ab218198) at 1/1000 dilution

  Lane 1: Mouse heart tissue lysate at 20 µg

  Lane 2: Mouse liver tissue lysate at 20 µg

  Lane 3: Rat heart tissue lysate at 20 µg

  Lane 4: Rat liver tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 427 kDa

  Observed band size: 425 kDa