Human PXN (Paxillin) knockout A-431 cell lysate

• WB

Lab

Western blot - Human PXN (Paxillin) knockout A-431 cell lysate (AB261701)

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

Lane 2 : PXN knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate 20 ug

Lane 3 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate 20 ug

Lane 4 : PC3 (Human prostate adenocarcinoma cell line) whole cell lysate 20 ug

Lanes 1 - 4 : Merged signal (red and green). Green - ab23510 observed at 65 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab23510 was shown to recognize PXN in wild-type A-431 cells as signal was lost at the expected MW in PXN knockout cell line ab261892 (knockout cell lysate ab261701). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and PXN knockout samples were subjected to SDS-PAGE. ab23510 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Paxillin antibody [M107] (<a href='/ko/products/primary-antibodies/paxillin-antibody-m107-ab23510'>ab23510</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

PXN knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human PXN (Paxillin) knockout A-431 cell line (<a href='/ko/products/cell-lines/human-pxn-paxillin-knockout-a-431-cell-line-ab261892'>ab261892</a>)

Lane 3:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Lane 4:

PC3 (Human prostate adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 65 kDa

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• WB

Lab

Western blot - Human PXN (Paxillin) knockout A-431 cell lysate (AB261701)

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

Lane 2 : PXN knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate 20 ug

Lane 3 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate 20 ug

Lane 4 : PC3 (Human prostate adenocarcinoma cell line) whole cell lysate 20 ug

Lanes 1 - 4 : Merged signal (red and green). Green - ab32115 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32115 was shown to specifically react with PXN in wild-type A-431 cells as signal was lost in PXN knockout cell line ab261892 (knockout cell lysate ab261701). Wild-type and PXN knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. ab32115 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Paxillin antibody [E228] (<a href='/ko/products/primary-antibodies/paxillin-antibody-e228-ab32115'>ab32115</a>) at 1/10000 dilution

Lane 1:

Wild-type A431 whole cell lysate at 20 µg

Lane 2:

PXN knockout A431 whole cell lysate at 20 µg

Lane 2:

Western blot - Human PXN (Paxillin) knockout A-431 cell line (<a href='/ko/products/cell-lines/human-pxn-paxillin-knockout-a-431-cell-line-ab261892'>ab261892</a>)

Lane 3:

U-87 MG whole cell lysate at 20 µg

Lane 4:

PC-3 whole cell lysate at 20 µg

Predicted band size: 65 kDa

Observed band size: 65 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Human PXN (Paxillin) knockout A-431 cell lysate (AB261701)

Lane 1 : Wild-type A431 cell lysate 20 ug Lane 2 : PXN knockout A431 cell lysate 20 ug Anti-PXN antibody [Y113] (ab32084) 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, ab32084 was shown to bind specifically to PXN. A band was observed at 70 kDa in wild-type A431 cell lysates with no signal observed at this size in PXN knockout cell line ab261892 (knockout cell lysate ab261701). To generate this image, wild-type and PXN knockout A431 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-Paxillin antibody [Y113] (<a href='/ko/products/primary-antibodies/paxillin-antibody-y113-ab32084'>ab32084</a>) at 1/1000 dilution

Lane 2:

Western blot - Human PXN (Paxillin) knockout A-431 cell lysate (ab261701)

Lane 2:

Western blot - Human PXN (Paxillin) knockout A-431 cell line (<a href='/ko/products/cell-lines/human-pxn-paxillin-knockout-a-431-cell-line-ab261892'>ab261892</a>)

Predicted band size: 65 kDa

Observed band size: 70 kDa

false

• NGS

Lab

Next Generation Sequencing - Human PXN (Paxillin) knockout A-431 cell lysate (AB261701)

X = 7 bp deletion, 1 bp insertion