Fig. 4.
mTORL1460P and mTORE2419K show constitutive activity. (A–C) HEK293 cells were transfected with pCDNA3 (vector), AU1-mTOR (wt, E2419K, or L1460P), or FLAG-Rheb1 (wt or N153T). To detect the phosphorylation of S6K or 4E-BP1, FLAG-S6K or FLAG-4E-BP1 was cotransfected. Cells then were serum-starved overnight and cultured in PBS for 1 h. The protein levels were detected by anti-FLAG (S6K, 4E-BP1, and Rheb), anti-AU-1 (mTOR), or anti-S6 antibody. The phosphorylation levels of S6K, 4E-BP1, and S6 were detected by phospho-specific antibodies. (D) HEK293 cells were transfected with AU1-mTOR (wt, E2419K, or L1460P). Cells were serum-starved overnight and then treated with rapamycin (100 nM). The expression and phosphorylation levels of S6 were detected by specific antibodies. wt, wild type.