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. 2010 Feb 12;5(2):e9197. doi: 10.1371/journal.pone.0009197

Figure 5. Cdc2 phosphorylation of raptor does not change mTORC1 complexes or signaling.

Figure 5

(A) HEK293T cells were transiently transfected with myc-raptor (wild-type or Ser696/Thr706/Ser711AAA: “3A”), AU1-mTOR, HA-GbL and Flag-PRAS40 for 16 hours, followed by 16 hours of nocodazole treatment following addition of fresh media to plates. Cells were lysed and myc-raptor was immunoprecipitated using an antibody against the myc-tag. Immunoprecipitates were resolved by SDS-PAGE and probed with antibodies against the Flag- and HA- tags. (B,C) HEK293T cells were transiently transfected with myc-raptor (wild-type or 3A) and Flag-S6K or Flag-4EBP1. 16 hours later, media was changed and nocodazole was added for 16 hours. Cells were lysed and lysated were split in two; myc-raptor was immunoprecipitated with an antibody against the myc-tag, and Flag-S6K or 4EBP1 were immunoprecipitated with an antibody against the Flag-tag. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. (D) Hela cells stably expressing myc-raptor (wt or 3A) with stable knockdown of endogenous raptor were treated with nocodazole for 16 hours, then Torin1 or roscovitine were added for 4 hours. Cells were lysed and myc-raptor was immunoprecipitated with an antibody against the myc-tag. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.