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

Figure 4. Cdc2 is the raptor Ser696, Thr706 kinase.

Figure 4

(A) Purified cdc2 can directly phosphorylate raptor on Thr706 and Ser696 in vitro. Myc-raptor (wild-type or S696/T706AA) was immunoprecipitated from hydroxyurea treated HEK293T cells. Immunoprecipitates were incubated with or without active recombinant Cdc2/cyclin B and immunoblotted with phospho-raptor Ser696, Thr706 or total raptor. (B) Endogenous raptor is phosphorylated on Thr706 in synchronized cells undergoing mitosis, and this phosphorylation is blocked by the CDK inhibitor roscovitine. A549 cells were synchronized by double thymidine block and endogenous raptor was immunoprecipitated at the indicated times after release with an anti-raptor antibody and immunoblotted with phospho-raptor Thr706. Whole cell lysates taken from the same cells were immunoblotted for mitotic markers phospho-histone H3 Ser10 and phospho-Plk1 Thr210. (C) Raptor immunoprecipitates with endogenous cyclin B. Hela cells stably expressing myc-wt raptor with stable knockdown of endogenous raptor treated with or without taxol for 16 hours. myc-tagged raptor was immunoprecipitated and immunoblotted for Cyclin B.