Role of the PIF‐binding pocket of PDK1 in the phosphorylation of S6K1, SGK1 and PKBα. Role of the PIF‐binding pocket of PDK1 in the phosphorylation of S6K1, SGK1 and PKBα. The indicated AGC substrates were incubated with wild‐type GST–PDK1 or mutant GST–PDK1[L155E] in the presence of magnesium and [γ‐32P]ATP as described in the Materials and methods. The activation of each AGC kinase was assessed by its ability to phosphorylate the peptide substrate Crosstide (GRPRTSSFAEG). Phosphorylation of the AGC kinase substrate was determined following electrophoresis on a 4–12% gradient polyacrylamide gel and the Coomassie Blue‐staining bands corresponding to each substrate were analysed on a phosphoimager. Phosphorylation was also analysed by subjecting the AGC kinases to immuno blotting with antibodies that specifically detect the T‐loop‐phosphorylated form of S6K1 (Thr252), SGK1 (Thr256) and PKBα (Thr308). Under the conditions used, phosphorylation of each substrate by PDK1 was linear with time and with the amount of enzyme added to the assay. Experiments were performed in duplicate using at least two separate time points. Duplicates within one experiment did not vary >10%, and usually <5%. Ricardo M. Biondi et al. EMBO J. 2001;20:4380-4390 © as stated in the article, figure or figure legend