ULK1 phosphorylates Ser389 of PDK1 linker region. (A) HA-ULK1 proteins were co-expressed with GST-PDK1 Kinase, C or PH proteins in HEK293T cells. The cell lysates were cleared and incubated with glutathione beads as described in Materials and Methods. Western blots for the precipitated proteins were obtained using anti-GST antibody (top panel). The anti-HA immunoblot (bottom panel) was also conducted using the same whole cell lysates. (B) HEK293T cells transfected with HA-ULK1 WT or KI were lysed and ULK1 assays were performed using GST-PDK1 linker protein as a substrate as described in Materials and Methods. The phosphorylated proteins were visualized by autoradiography (top panel). ULK1 expression was detected by the immunoblot obtained from the same whole cell lysates (bottom panel). The above results shown are representative of three independent experiments. (C) HEK293T cells were transfected with GST-PDK1 linker and HA-ULK1 WT. After 48 h of transfection, PDK1 linker proteins were pulled down and eluted as described in Materials and Methods. The fractions of eluted proteins were subjected to SDS-PAGE and the gel was stained by Coomassie R250 solution. (D) Phosphorylated proteins from (C) were analyzed by mass spectrometry as described in Materials and Methods.