Fig. 3

PDHA phospho-site mutants decrease enzymatic activity of PDC. Pyruvate dehydrogenase enzymatic activity microplate immunocapture assay (abcam ab110671) was used to quantify the PDC activity in MiaPaca2 and phospho-site mutant cells after culture for 16 h in 21% or 1% oxygen. Each sample was normalized to captured PDHA1 as determined by western blot of captured protein. A Kinetic readings of parental MiaPaca2, wild-type PDHA1 re-expressed, and S-232,293,300-A (AAA) PDHA1 re-expressed. Results are the average of 3 experiments, and PDHA KO cells had 0.00 absorbance (data not shown). N = 3 biological replicates. B Endpoint measures for curves reported in A with statistical significance calculated by T test. C Focus on the significance of serine 232 with analysis of PDHA1 mutants S232A (ASS) and S293A/S300A (SAA). These two mutants show similar activity to the S232A/S293A/S300A mutant assayed A and B. However, the enzymes are still insensitive to hypoxia. N = 3, P=NS. D Endpoint measures for curves reported in C with statistical significance calculated by T test. Error bars, s.e.m. *P< 0.05, **P< 0.01