Anti-GAPDH antibody [6C5] - Loading Control

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

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 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 (ab150117) (shown in green). Nuclear DNA was labeled in blue with DAPI.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

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 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 (ab150117) at 2 μg/ml (shown in green) and 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

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 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 (ab150117) (shown in green). Nuclear DNA was labeled in blue with DAPI.

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

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker ab40839 staining at 1/5000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 76 kDa in Wild-type A549 cell lysates with no signal observed at this size in EZR 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

Lanes 1 - 3:

Western blot - Anti-Ezrin antibody [EP886Y] - Plasma Membrane Marker (<a href='/en-us/products/primary-antibodies/ezrin-antibody-ep886y-plasma-membrane-marker-ab40839'>ab40839</a>) at 1/5000 dilution

Lanes 1 - 3:

Western blot - Anti-Ezrin antibody [EP886Y] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/ezrin-antibody-ep886y-bsa-and-azide-free-ab239832'>ab239832</a>) at 1/5000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human EZR knockout A549 cell line (<a href='/en-us/products/cell-lines/human-ezr-knockout-a549-cell-line-ab300934'>ab300934</a>) at 20 µg

Lane 3:

HeLa at 20 µg

Secondary

All lanes:

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

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-Fibroblast activation protein, alpha antibody [EPR20021] ab207178 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 80-100 kDa. 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Fibroblast activation protein, alpha antibody [EPR20021] (<a href='/en-us/products/primary-antibodies/fibroblast-activation-protein-alpha-antibody-epr20021-ab207178'>ab207178</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 BFA (5 g/mL, 6h) at 20 µg

Lane 2:

Wild-type MCF7 BFA (0 g/mL, 6h) at 20 µg

Lane 3:

FAP knockout MCF7 BFA (5 g/mL, 6 h) at 20 µg

Lane 4:

FAP knockout MCF7 BFA (0 g/mL, 6 h) at 20 µg

Secondary

All lanes:

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

Predicted band size: 80-100 kDa

Observed band size: 80-100 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EP206Y] to PYK2 ab81266 staining at 1/2500 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta.

A band was observed at 120 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in PTK2B knockout HCT 116 cell line (ab301190). We are unable to identify the non-specific bands at 75kDa and 40kDa.

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-PYK2 antibody [EP206Y] (<a href='/en-us/products/primary-antibodies/pyk2-antibody-ep206y-ab81266'>ab81266</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human PTK2B knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ptk2b-knockout-hct116-cell-line-ab301190'>ab301190</a>) at 20 µg

Lane 3:

Wild-type A549 at 20 µg

Lane 4:

PTK2B knockout A549 at 20 µg

Secondary

All lanes:

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

Predicted band size: 116 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[YE353] to PYK2 ab32571 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 120 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in PTK2B knockout HCT 116 cell line (ab301190).

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-PYK2 antibody [YE353] (<a href='/en-us/products/primary-antibodies/pyk2-antibody-ye353-ab32571'>ab32571</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human PTK2B knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ptk2b-knockout-hct116-cell-line-ab301190'>ab301190</a>) at 20 µg

Lane 3:

Wild-type A549 at 20 µg

Lane 4:

PTK2B knockout A549 at 20 µg

Secondary

All lanes:

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

Predicted band size: 116 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-FKBP52 antibody [EPR21120] ab230952 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 52 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in FKBP4 knockout HCT 116 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-FKBP52 antibody [EPR21120] (<a href='/en-us/products/primary-antibodies/fkbp52-antibody-epr21120-ab230952'>ab230952</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human FKBP4 knockout HCT116 cell line (ab300946) at 20 µg

Lane 3:

LNCaP at 20 µg

Lane 4:

THP-1 at 20 µg

Secondary

All lanes:

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

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-SHP1 antibody [EPR5519] ab124942 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 68 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in PTPN6 CRISPR-Cas9 Edited 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-SHP1 antibody [EPR5519] (<a href='/en-us/products/primary-antibodies/shp1-antibody-epr5519-ab124942'>ab124942</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human PTPN6 knockout MCF7 cell line (ab287732) at 20 µg

Lane 3:

THP-1 at 20 µg

Lane 4:

SH-SY5Y at 20 µg

Secondary

All lanes:

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

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-SHP1 antibody [Y476] ab32559 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 68 kDa in Wild-type MCF7 cell lysates with a band observed at 55 kDa in PTPN6 CRISPR-Cas9 Edited 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-SHP1 antibody [Y476] (<a href='/en-us/products/primary-antibodies/shp1-antibody-y476-ab32559'>ab32559</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human PTPN6 knockout MCF7 cell line (ab287732) at 20 µg

Lane 3:

THP-1 at 20 µg

Lane 4:

SH-SY5Y at 20 µg

Secondary

All lanes:

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

Predicted band size: 68 kDa

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-FKBP52 antibody [EPR6619] ab124906 staining at 1/50000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 52 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in FKBP4 knockout HCT 116 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-FKBP52 antibody [EPR6619] (<a href='/en-us/products/primary-antibodies/fkbp52-antibody-epr6619-ab124906'>ab124906</a>) at 1/50000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human FKBP4 knockout HCT116 cell line (ab300946) at 20 µg

Lane 3:

LNCaP at 20 µg

Lane 4:

THP-1 at 20 µg

Secondary

All lanes:

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

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal [EP206Y] to PYK2 ab81266 staining at 1/2000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 116 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in PTK2B knockout HCT 116 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-PYK2 antibody [EP206Y] (<a href='/en-us/products/primary-antibodies/pyk2-antibody-ep206y-ab81266'>ab81266</a>) at 1/2000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human PTK2B knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ptk2b-knockout-hct116-cell-line-ab301190'>ab301190</a>) at 20 µg

Lane 3:

A549 at 20 µg

Secondary

All lanes:

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

Predicted band size: 116 kDa

Observed band size: 116 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-AKT2 antibody [EP1676] ab131168 staining at 1/5000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 56 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in AKT2 knockout MCF7 cell line. This antibody also detects AKT1; gel running protocol should be optimised to separate these bands. 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-AKT2 antibody [EP1676] (<a href='/en-us/products/primary-antibodies/akt2-antibody-ep1676-ab131168'>ab131168</a>) at 1/5000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human AKT2 knockout MCF7 cell line (ab286253) at 20 µg

Lane 3:

A549 at 20 µg

Lane 4:

THP-1 at 20 µg

Secondary

All lanes:

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

Predicted band size: 56 kDa

Observed band size: 56 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-FKBP52 antibody [EPR6618] ab129097 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 52 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in FKBP4 knockout HCT 116 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-FKBP52 antibody [EPR6618] (<a href='/en-us/products/primary-antibodies/fkbp52-antibody-epr6618-ab129097'>ab129097</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human FKBP4 knockout HCT116 cell line (ab300946) at 20 µg

Lane 3:

LNCaP at 20 µg

Lane 4:

THP-1 at 20 µg

Secondary

All lanes:

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

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EPR25933-111] to Cystatin SN/CST1 ab307416 staining at 1/1000 dilution; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 14 kDa. 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) and Goat anti-Mouse 680RD at 1/20,000 dilution. This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-Cystatin SN/CST1 antibody [EPR25933-111] (<a href='/en-us/products/primary-antibodies/cystatin-sn-cst1-antibody-epr25933-111-ab307416'>ab307416</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 BFA (5g/mL,6h) at 20 µg

Lane 2:

Wild-type A549 BFA (0 g/mL,6h) at 20 µg

Lane 3:

CST1 knockout A549 BFA (5 g/mL, 6 h) at 20 µg

Lane 4:

CST1 knockout A549 BFA (0 g/mL, 6 h) at 20 µg

Lane 5:

SW480 at 20 µg

Lane 6:

HCT 116 at 20 µg

Secondary

All lanes:

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

Predicted band size: 14 kDa

Observed band size: 14 kDa

true

Exposure time: 5s

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

This data was developed using the same antibody clone in a different buffer formulation (ab51067).

Anti-MET antibody [EP1454Y] (ab51067) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab51067 was shown to bind specifically to MET. A band was observed at 40-50 kDa in in wild-type HeLa cell lysates with no signal observed at this size in MET knockout cell line. To generate this image, wild-type and MET 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Met (c-Met) antibody [EP1454Y] - N-terminal (<a href='/en-us/products/primary-antibodies/met-c-met-antibody-ep1454y-n-terminal-ab51067'>ab51067</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MET (Met (c-Met)) knockout HeLa cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

T-47D 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

Observed band size: 40-50 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-SPG3A/ATL1 antibody [EPR28057-64] ab316111 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 64 kDa in Wild-type A549 cell lysates with no signal observed at this size in ATL1 knockout A549 cell line (ab326073). 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-SPG3A/ATL1 antibody [EPR28057-64] (<a href='/en-us/products/primary-antibodies/spg3a-atl1-antibody-epr28057-64-ab316111'>ab316111</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human ATL1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-atl1-knockout-a549-cell-line-ab326073'>ab326073</a>) at 20 µg

Lane 3:

SH-SY5Y at 20 µg

Lane 4:

HepG2 at 20 µg

Secondary

All lanes:

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

Predicted band size: 64 kDa

Observed band size: 64 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-ATG7 antibody [EP1759Y] ab52472 staining at 1/2000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 78 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in ATG7 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-ATG7 antibody [EP1759Y] (<a href='/en-us/products/primary-antibodies/atg7-antibody-ep1759y-ab52472'>ab52472</a>) at 1/2000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

ATG7 knockout U-87 MG at 20 µg

Lane 3:

THP-1 at 20 µg

Lane 4:

Jurkat at 20 µg

Secondary

All lanes:

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

Predicted band size: 78 kDa

Observed band size: 78 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-MUC13 antibody [EPR21901] ab235450 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 100-150 kDa in Wild-type A549 cell lysates with no signal observed at this size in MUC13 knockout A549 cell line. Two close non-specific bands were detected near to MUC13. We have not invesitgated the identity of these bands. 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MUC13 antibody [EPR21901] (<a href='/en-us/products/primary-antibodies/muc13-antibody-epr21901-ab235450'>ab235450</a>) at 1/100 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

MUC13 knockout A549 at 20 µg

Secondary

All lanes:

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

Predicted band size: 100-150 kDa

Observed band size: 100-150 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-c-Kit antibody [EPR25707-134] ab283653 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 140 kDa in Wild-type HAP1 cell lysates with no signal observed at this size in KIT knockout HAP1 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-c-Kit antibody [EPR25707-134] (<a href='/en-us/products/primary-antibodies/c-kit-antibody-epr25707-134-ab283653'>ab283653</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 at 20 µg

Lane 2:

KIT knockout HAP1 at 20 µg

Lane 3:

HUVEC at 20 µg

Lane 4:

Jurkat at 20 µg

Secondary

All lanes:

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

Predicted band size: 140 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Anti-CHCHD10 antibody produced in rabbit HPA003440 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 kDa in Wild-type A549 ab288558 cell lysates with no signal observed at this size in CHCHD10 knockout A549 cell line (ab326017). 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:

Anti-CHCHD10 antibody produced in rabbit at 1/1000 dilution

Lane 1:

Wild-type A549 ab288558 at 20 µg

Lane 2:

Western blot - Human CHCHD10 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-chchd10-knockout-a549-cell-line-ab326017'>ab326017</a>) at 20 µg

Lane 3:

LNCaP at 20 µg

Lane 4:

U-2 OS at 20 µg

Secondary

All lanes:

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

Predicted band size: 14 kDa

Observed band size: 18 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-SEMA3B antibody (ab48197) staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab48197 was shown to bind specifically to SEMA3B. A band was observed at 81 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in SEMA3B knockout cell line (ab326024). Arrow indicates the target band.To generate this image, wild-type and SEMA3B 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-SEMA3B antibody (<a href='/en-us/products/primary-antibodies/sema3b-antibody-ab48197'>ab48197</a>) at 1/5000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human SEMA3B knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-sema3b-knockout-hct116-cell-line-ab326024'>ab326024</a>) at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

RKO cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 81 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[E343] to S6K1 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 (ab326023). 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] (<a href='/en-us/products/primary-antibodies/s6k1-antibody-e343-ab32529'>ab32529</a>) at 1/10000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human RPS6KB1 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-rps6kb1-knockout-u-87-mg-cell-line-ab326023'>ab326023</a>) 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

Predicted band size: 59 kDa

Observed band size: 59 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[E175] to S6K1 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 (ab326023). 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] (<a href='/en-us/products/primary-antibodies/s6k1-antibody-e175-ab32359'>ab32359</a>) at 1/5000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human RPS6KB1 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-rps6kb1-knockout-u-87-mg-cell-line-ab326023'>ab326023</a>) 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

Predicted band size: 59 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EPR27133-30] to RNF5/NG2 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 (ab326022). 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] (<a href='/en-us/products/primary-antibodies/rnf5-ng2-antibody-epr27133-30-ab308066'>ab308066</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human RNF5 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-rnf5-knockout-a549-cell-line-ab326022'>ab326022</a>) 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

Predicted band size: 20 kDa

Observed band size: 18 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EPR25155-48] to PICK1 ab290727 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 (ab326021). 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 [EPR25155-48] (<a href='/en-us/products/primary-antibodies/pick1-antibody-epr25155-48-ab290727'>ab290727</a>) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human PICK1 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-pick1-knockout-u-87-mg-cell-line-ab326021'>ab326021</a>) 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

Predicted band size: 47 kDa

Observed band size: 47 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EPR4130(3)] to PICK1 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 (ab326021). 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)] (<a href='/en-us/products/primary-antibodies/pick1-antibody-epr41303-ab133773'>ab133773</a>) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human PICK1 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-pick1-knockout-u-87-mg-cell-line-ab326021'>ab326021</a>) 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

Predicted band size: 47 kDa

Observed band size: 47 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[HL1227] to Artemis ab313837 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 80-90 kDa in Wild-type A549 cell lysates with no signal observed at this size in DCLRE1C knockout A549 cell line (ab326018).

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-Artemis antibody [HL1227] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/artemis-antibody-hl1227-bsa-and-azide-free-ab313837'>ab313837</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human DCLRE1C knockout A549 cell line (<a href='/en-us/products/cell-lines/human-dclre1c-knockout-a549-cell-line-ab326018'>ab326018</a>) at 20 µg

Lane 3:

Jurkat at 20 µg

Lane 4:

PC-3 at 20 µg

Secondary

All lanes:

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

Predicted band size: 100 kDa

Observed band size: 80-90 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EPR23090-181] to Acid sphingomyelinase ab272729 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 74 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in SMPD1 knockout MCF7 cell line (ab326026). To generate this image, 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (<a href='/en-us/products/primary-antibodies/acid-sphingomyelinase-antibody-epr23090-181-ab272729'>ab272729</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human SMPD1 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-smpd1-knockout-mcf7-cell-line-ab326026'>ab326026</a>) at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

Ramos at 20 µg

Secondary

All lanes:

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

Predicted band size: 70 kDa

Observed band size: 74 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

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 (ab326025). 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:

Western blot - Human SLC11A2 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-slc11a2-knockout-a549-cell-line-ab326025'>ab326025</a>) at 20 µg

Secondary

All lanes:

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

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-SEMA3B antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, anti-SEMA3B antibody was shown to bind specifically to SEMA3B. A band was observed at 83 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in SEMA3B knockout cell line (ab326024). To generate this image, wild-type and SEMA3B 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Anti-SEMA3B antibody at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human SEMA3B knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-sema3b-knockout-hct116-cell-line-ab326024'>ab326024</a>) at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

RKO 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

Observed band size: 83 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-Ezrin antibody [EPR23353-55] ab270442 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 76 kDa in Wild-type A549 cell lysates with no signal observed at this size in EZR 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Ezrin antibody [EPR23353-55] (<a href='/en-us/products/primary-antibodies/ezrin-antibody-epr23353-55-ab270442'>ab270442</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human EZR knockout A549 cell line (<a href='/en-us/products/cell-lines/human-ezr-knockout-a549-cell-line-ab300934'>ab300934</a>) at 20 µg

Lane 3:

HeLa at 20 µg

Secondary

All lanes:

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

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-AKT1 + AKT2 antibody [EPR17062] ab182729 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 56, 58 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in AKT2 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 BSA 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-AKT1 + AKT2 antibody [EPR17062] (<a href='/en-us/products/primary-antibodies/akt1-akt2-antibody-epr17062-ab182729'>ab182729</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human AKT2 knockout MCF7 cell line (ab286253) at 20 µg

Lane 3:

A549 at 20 µg

Lane 4:

THP-1 at 20 µg

Secondary

All lanes:

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

Predicted band size: 56 kDa,58 kDa

Observed band size: 56 kDa,58 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Anti-MET antibody [EPR19554-110] (ab254252) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab254252 was shown to bind specifically to MET. A band was observed at 80-140 kDa in wild-type HeLa cell lysates with no signal observed at this size in MET knockout cell line. To generate this image, wild-type and MET 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution. The full length MET protein is glycosylated and has multiple cleavage sites, so cleaved products can be detected between ~40-140 kDa

All lanes:

Western blot - Anti-Met (c-Met) antibody [EPR19554-110] (<a href='/en-us/products/primary-antibodies/met-c-met-antibody-epr19554-110-ab254252'>ab254252</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MET (Met (c-Met)) knockout HeLa cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

T-47D 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

Observed band size: 80-140 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

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, 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 ab273712 (knockout 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-beta Catenin antibody [IGX4794R-3] (<a href='/en-us/products/primary-antibodies/beta-catenin-antibody-igx4794r-3-ab223075'>ab223075</a>) 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 (<a href='/en-us/products/cell-lysates/human-ctnnb1-beta-catenin-knockout-hct116-cell-lysate-ab275247'>ab275247</a>)

Lane 2:

Western blot - Human CTNNB1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ctnnb1-knockout-hct116-cell-line-ab273712'>ab273712</a>)

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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

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 - ab223075 observed at 95 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
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 ab273712 (CTNNB1 knockout 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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] (<a href='/en-us/products/primary-antibodies/beta-catenin-antibody-igx4794r-3-ab223075'>ab223075</a>) 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 (<a href='/en-us/products/cell-lysates/human-ctnnb1-beta-catenin-knockout-hct116-cell-lysate-ab275247'>ab275247</a>)

Lane 2:

Western blot - Human CTNNB1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ctnnb1-knockout-hct116-cell-line-ab273712'>ab273712</a>)

Lane 2:

CTNNB1 knockout HCT116 cell lysate at 20 µg

Predicted band size: 85 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Rabbit Monoclonal[EPR5108] to HINT1 ab124912 staining at 1/500 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 14 kDa in Wild-type HeLa cell lysates with no signal observed at this size in HINT1 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 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-HINT1 antibody [EPR5108] (<a href='/en-us/products/primary-antibodies/hint1-antibody-epr5108-ab124912'>ab124912</a>) at 1/500 dilution

Lane 1:

Wild-type HeLa at 20 µg

Lane 2:

Western blot - Human HINT1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-hint1-knockout-hela-cell-line-ab265776'>ab265776</a>) at 20 µg

Lane 3:

Jurkat at 20 µg

Lane 4:

HeLa Membrane at 20 µg

Lane 5:

EMPTY

Lane 6:

Western blot - Recombinant Human HINT1 protein (Tag Free) (<a href='/en-us/products/proteins-peptides/recombinant-human-hint1-protein-ab87362'>ab87362</a>) at 0.1 µg

Secondary

Lanes 1 - 6:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 6:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa,37 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Performed under reducing conditions.

Samples are non-boiled as boiling may cause protein aggregates.

False colour image of Western blot : Anti-CD47 antibody [EPR24922-21] (ab300124) 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, ab300124 was shown to bind specifically to CD47. A diffuse band was observed at 47-52 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in CD47 knockout cell line ab266324 (knockout cell lysate ab257220).

To generate this image, wild-type and CD47 knockout HEK-293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Negative control : HepG2 (PMID : 28378740).

All lanes:

Western blot - Anti-CD47 antibody [EPR24922-21] (<a href='/en-us/products/primary-antibodies/cd47-antibody-epr24922-21-ab300124'>ab300124</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

CD47 knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Observed band size: 47-52 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-LATS1 antibody [EPR23057-116] (ab243656) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab243656 was shown to bind specifically to LATS1. A band was observed at 127 kDa in wild-type THP-1 cell lysates with no signal observed at this size in LATS1 knockout cell line. To generate this image, wild-type and LATS1 knockout THP-1 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-LATS1/WARTS antibody [EPR23057-116] (<a href='/en-us/products/primary-antibodies/lats1-warts-antibody-epr23057-116-ab243656'>ab243656</a>) at 1/2000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human LATS1 (WARTS) knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-lats1-warts-knockout-thp-1-cell-line-ab277862'>ab277862</a>)

Lane 2:

LATS1 knockout THP-1 cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

PC-3 cell lysate at 20 µg

Lane 5:

HeLa 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

Predicted band size: 126 kDa

Observed band size: 127 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-TAF15 antibody [EPR9197(B)] ab134916 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 74 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in TAF15 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-TAF15 antibody [EPR9197(B)] (<a href='/en-us/products/primary-antibodies/taf15-antibody-epr9197b-ab134916'>ab134916</a>) at 1/10000 dilution

Lane 1:

Wild-type U-87 MG whole cell lysate at 20 µg

Lane 2:

TAF15 knockout U-87 MG whole cell lysate at 20 µg

Lane 3:

Raji whole cell lysate at 20 µg

Lane 4:

HT1080 whole cell lysate at 20 µg

Lane 5:

PC-3 whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 62 kDa

Observed band size: 74 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [6C5] - Loading Control (AB8245)

Western blot : Anti-S100A11 antibody [EPR11171(B)] ab169530 staining at 1/1000 dilution; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 12 kDa in Wild-type HeLa cell lysates with no signal observed at this size in S100A11 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 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) and Goat anti-Mouse 680RD at 1/20,000 dilution. This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-S100A11 antibody [EPR11171(B)] (<a href='/en-us/products/primary-antibodies/s100a11-antibody-epr11171b-ab169530'>ab169530</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa at 20 µg

Lane 2:

Western blot - Human S100A11 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-s100a11-knockout-hela-cell-line-ab265085'>ab265085</a>) at 20 µg

Lane 3:

U-2 OS at 20 µg

Lane 4:

PC-3 at 20 µg

Secondary

All lanes:

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

Predicted band size: 12 kDa

Observed band size: 12 kDa

true

Exposure time: 10s