FIGURE 7.

Raptor T908A mutation does not significantly alter binding of Raptor to mTOR and S6K1. A, HEK293E cells were cotransfected with Myc- or AU1-tagged mTOR and FLAG-tagged Raptor (WT or T908A), serum-starved, and stimulated with 100 nm insulin for 30 min. Myc-mTOR or AU1-mTOR was immunoprecipitated (IP) from the whole cell extract using anti-Myc or anti-AU1 antibodies, respectively. The level of FLAG-Raptor associated with either Myc-mTOR or AU1-mTOR immunoprecipitates was determined by immunoblotting (IB) with anti-FLAG. Total amounts of FLAG-Raptor, Myc-mTOR, and AU1-mTOR from the whole cell extract were indicated on Western blot analysis by using anti-FLAG, anti-Myc, and anti-AU1 antibodies, respectively. B, HEK293E cells were cotransfected with AU1-mTOR, FLAG-Raptor (WT or T908A), and HA-S6K1; serum-starved; and stimulated with 100 nm insulin for 30 min. FLAG-Raptor was immunoprecipitated from the whole cell extract by anti-FLAG, and the level of HA-S6K1 associated with the FLAG-Raptor immune complex was assessed on Western blot analysis using anti-HA. Total signal inputs of S6K1 and Raptor from the whole cell extract were also indicated on Western blot analysis. Insulin effect was indicated by the increase of phospho-ERK1/2 signals in the whole cell lysate.