Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PDK1 knockout HAP1 cell lysate (20 µg)
Lane 3: Human fetal heart lysate (20 µg)
Lane 4: LnCaP cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab110335 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab110335 detected the expected band for PDK1 in wild-type cells and the band was not seen in knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PDK1 knockout samples were subjected to SDS-PAGE. ab110335 and ab8245 (loading control to GAPDH) were diluted 1 μg/mL and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PDK1 antibody [2H3AA11] (ab110335) at 1 µg/ml

Lane 1 : Human heart lysate at 10 µg
Lane 2 : Human liver lysate 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: 49 kDa

All lanes : Anti-PDK1 antibody [2H3AA11] (ab110335) at 1 µg/ml

Lane 1 : HeLa cells incubated for 24 hours with vehicle
Lane 2 : HeLa cells incubated for 24 hours with 100 µM cobalt chloride (CoCl2)
Lane 3 : HeLa cells incubated for 24 hours with 1 mM deferoxamine (DFO)

Lysates/proteins at 30 µg per lane.

Predicted band size: 49 kDa



Treatment with either CoCl2 or DFO results in increased expression of PDK1, which is known to be transcriptionally upregulated in response to hypoxia.

HL-60 cells were stained with 1 µg/mL PDK1 antibody (ab110335) (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.

Immunocytochemistry image of PDK1 stained HeLa cells. The Upper image shows cells which were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with the PDK1 antibody (ab110335) at 2 µg/mL for 2 hours at room temperature or over night at 4°C. The secondary antibody was (green) Alexa Fluor® 488 goat anti-mouse IgG (H+L) at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. The target protein locates to the mitochondrial matrix.
The lower image shows cells co-stained with an antibody against HSP60 (red), an enzyme also located in the mitochondrial matrix. The composite image shows an identical mitochondrial pattern for both antibodies indicated by merged orange color.