Human PDK4 knockout HeLa cell line

• WB

Lab

Western blot - Human PDK4 knockout HeLa cell line (AB261805)

Lanes 1-3 : Merged signal (red and green). Green - ab110336 observed at 48 kDa. Red - loading control, ab129002 observed at 124 kDa.

ab110336 Anti-PDK4 antibody [1C2BG5] was shown to specifically react with PDK4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261805 (knockout cell lysate ab257217) was used. Wild-type and PDK4 knockout samples were subjected to SDS-PAGE. ab110336 and Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 1000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PDK4 antibody [1C2BG5] (<a href='/ko/products/primary-antibodies/pdk4-antibody-1c2bg5-ab110336'>ab110336</a>) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PDK4 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PDK4 knockout HeLa cell line (ab261805)

Lane 3:

Human heart tissue lysate at 20 µg

Predicted band size: 46 kDa

Observed band size: 48 kDa

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• WB

Lab

Western blot - Human PDK4 knockout HeLa cell line (AB261805)

Western blot : Anti-PDK4 antibody [1C2BG5] (ab110336) staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab110336 was shown to bind specifically to PDK4. A band was observed at 45 kDa in wild-type HeLa cell lysates with no signal observed at this size in PDK4 knockout cell line. To generate this image, wild-type and PDK4 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PDK4 antibody [1C2BG5] (<a href='/ko/products/primary-antibodies/pdk4-antibody-1c2bg5-ab110336'>ab110336</a>) at 1 µg/mL

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PDK4 knockout HeLa cell lysate at 20 µg

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

THP-1 cell lysate at 20 µg

Lane 5:

Jurkat cell lysate at 20 µg

Lane 6:

A431 cell lysate at 20 µg

Lane 7:

Human Heart cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 45 kDa

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• Sanger seq

Unknown

Sanger Sequencing - Human PDK4 knockout HeLa cell line (AB261805)

Allele-2 : 1 bp insertion in exon 1.

• Sanger seq

Unknown

Sanger Sequencing - Human PDK4 knockout HeLa cell line (AB261805)

Allele-1 : 1 bp deletion in exon 1.