Anti-PDK4 antibody [1C2BG5]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PDK4 antibody [1C2BG5] (AB110336)

ICC/IF image of ab110336 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab110336, 10μg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PDK4 antibody [1C2BG5] (AB110336)

Overlay histogram showing HepG2 cells stained with ab110336 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab110336, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• WB

Lab

Western blot - Anti-PDK4 antibody [1C2BG5] (AB110336)

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] (ab110336) 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 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 - Anti-PDK4 antibody [1C2BG5] (AB110336)

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] (ab110336) 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 (<a href='/en-us/products/cell-lines/human-pdk4-knockout-hela-cell-line-ab261805'>ab261805</a>)

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 - Anti-PDK4 antibody [1C2BG5] (AB110336)

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] (ab110336) 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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• WB

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Western blot - Anti-PDK4 antibody [1C2BG5] (AB110336)

All lanes:

Western blot - Anti-PDK4 antibody [1C2BG5] (ab110336) at 1 µg/mL

Lane 1:

HepG2 whole cell lysate at 10 µg

Lane 2:

Human heart tissue at 10 µg

Lane 3:

Recombinant Human PDK1 at 0.01 µg

Lane 4:

Recombinant Human PDK2 at 0.01 µg

Lane 5:

Recombinant Human PDK3 at 0.01 µg

Lane 6:

Recombinant Human PDK4 at 0.01 µg

Predicted band size: 46 kDa

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• WB

CiteAb

Western blot - Anti-PDK4 antibody [1C2BG5] (AB110336)

PDK4 western blot using anti-PDK4 antibody [1C2BG5] ab110336. Publication image and figure legend from Sun, S., Liu, J., et al., 2017, Cell Death Dis, PubMed 28594398.

ab110336 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab110336 please see the product overview.

FAM210B knockdown cells show decreased PDK4 expression. (a) Heat map of the top 25 differentially regulated genes in siFAM210B and negative control A549 cells. (b) Quantitative PCR analyses of the relative expression of the indicated genes in siFAM210B normalized to negative control SKOV3 cells. (c) Immunoblot for PDK4 in the indicated treated SKOV3 cells. (d) Schematically depicted PDK function in SKOV3 cell metabolism. (e) Western blot analysis of the phosphorylation levels of PDH-E1α in the indicated treated SKOV3 cells. (f) Normalized OCR and (g) normalized ECAR in siPDK4 (n=6) and negative control SKOV3 cells (n=8 wells)

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