Figure 3.

eIF4B Ser422 is dephosphorylated in PDK1 null and PDK1 PIF pocket mutant ES cells. Wt and PDK1^−/− knockout (A) or PDK1 PIF pocket mutant (B) ES cells were starved for 16–18 h in the presence or absence of 20 nM rapamycin and then stimulated with 20% serum for 15 min. Total cell extracts were resolved by SDS–PAGE and proteins were detected by immunoblotting using phospho-eIF4B S422, phospho-S6K1 T389, phospho-S6 S240/244, and phospho-ERK1/2 T202/Y204 antibodies. Membranes were reprobed with antibodies against the indicated total proteins and against PDK1 (arrows on the right indicate nonspecific bands).