ULK1 interacts with PDK1 and induces phosphorylation. (A) HEK293T cells were transfected with HA-ULK1 and Myc-PDK1 constructs. After 48 h of transfection, the cells were lysed, and the lysates were subjected to immunoprecipitation using anti-Myc antibody. The immune complexes were washed and the immunoblots for PDK1 were performed as described in Materials and Methods (upper panel). The same cell lysates were used for anti-HA immunoblot (lower panel). (B) HA-ULK1 was expressed in HEK293T cells. Whole cell lysate was analyzed by immunoblotting for the indicated proteins. (C) HEK293T cells transfected with Myc-PDK1 and HA-ULK1 wild-type (WT) or kinase-inactive (KI) mutant were lysed and subject to immunoprecipitation with anti-Myc antibody. The immune complexes were incubated with λ phosphatase, washed, and subjected to anti-Myc immunoblot as described in Materials and Methods (top panel). Anti-Myc and anti-HA immunoblots were conducted using the same cell lysates (middle and bottom panels, respectively). (D) HA-ULK1 and GST-PDK1 were expressed in the HEK293T cells. The cell lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoblots were obtained with the indicated antibodies. (E) The scheme of PDK1 truncation mutants. (F) HEK293T cells transfected with HA-ULK1 and GST-PDK1 WT, kinase and linker (KL), C-terminal (C) or PH domain (PH) constructs were lysed, and the lysates were subjected to co-immunoprecipitation. The results shown are representative of three independent experiments.