ULK1 phosphorylates Ser389 of PDK1. (A) HEK293T cells were transfected with the indicated constructs. After 48 h of transfection, the cells were lysed and the lysates were subjected to immunoprecipitation using anti-Myc antibody (top panel). ULK1 immunoblot was performed using the same cell lysates (bottom panel). (B) Myc-PDK1 and Flag-ULK1 were overexpressed in the HEK293T cells. Immunoprecipitated Myc-PDK1 proteins were probed with an anti-pS389 PDK1 antibody (top panel). (C) HA-ULK1 WT and KI were overexpressed in the HEK293T cells and endogenous PDK1 proteins were immunoprecipitated using the anti-PDK1 antibody (top and middle panel). ULK1 expression was detected in the same cell lysates (bottom panel). (D) After the addition of 5 μM Oligomycin A to HEK293T cells at the indicated time points, the cells were lysed for immunoprecipitation of PDK1. The immune complexes were subjected to western blot using anti-pS389 and anti-PDK1 antibodies (top and bottom panel, respectively) (E) HEK293T cells were treated with 1 μM rapamycin at the indicated time points. Immunoprecipitated PDK1 proteins were probed with anti-pS389 and anti-PDK1 antibodies (top and upper middle panels, respectively). AP indicates alkaline phosphatase blotting. (F) HEK293T cells were treated with ULK1 inhibitor (1 μM, SBI-0206965) for 20 h, followed by addition of oligomycin A (5 μM) or DMSO for 4 h. Immunoprecipitated proteins using PDK1 antibody were analyzed via immunoblotting using the indicated antibodies. The above results shown are representative of three independent experiments.