Phosphorylation of PDK1 Ser389 regulates substrate phosphorylation. (A) Flag-PDK1 WT and S389A proteins were purified from HAP1 stable cells as described in Materials and Methods. PDK1 kinase activity was measured using ADP-GloTM PDK1 kinase assay kit according to the manufacturer’s instructions. The data represent means ± SD of three experiments. Statistical analysis was performed using Student’s t-test, and P-value < 0.05 was considered significant; however, the calculated P value was 0.69. n.s.: not significant (B) HAP1 PDK1 knockout cells stably expressing PDK1 WT and S389A mutant were lysed and the whole cell lysates were analyzed by immunoblotting using the indicated antibodies. (C) Negative feedback loop model between PDK1 and ULK1.