Fig. 3.

mTOR modulates S371 phosphorylation by regulating phosphatase activity. Data are representative of at least three independent experiments. (A) Schematic representation of the WT and mutant forms of S6K1. (B) HEK293 cells were transfected with WT and C terminus–truncated S6K1 mutants and lysed. After immunoprecipitation, immunoblot analysis was performed. (C) HEK293 cells were transfected with WT or mutant S6K1, lysed, immunoprecipitated, and analyzed by immunoblot. (D) HEK293 cells were pretreated with DMSO or rapamycin (20 nM) for 1 h, and then transfected with WT or mutant S6K1 for 16 h. After cell lysis, immunoprecipitation and immunoblot analyses were performed. (E) Mouse fibroblasts were first treated with rapamycin for 2 h, and then treated with DMSO or okadaic acid (200 nM) for an additional 2 h. Cells were lysed and then analyzed by immunoblot. (F) Stable PP2A A subunit knockdown mouse fibroblasts were generated using shRNAs. Cells were treated with 5 nM rapamycin for 3 h, lysed, and analyzed by immunoblot analysis.