Figure 2. Rab1A is essential for mTORC1 activation by AA in human cells.
(A) HEK293E cells infected with lentiviral Rab1A shRNAs were analyzed for P-S6K1(T389), S6K, P-AKT(S473), AKT, and PARP cleavage.
(B) HEK293E cells infected with Rab1A shRNA were starve and re-stimulated with AA for 10 min, and analyzed for P-S6K1(T389).
(C) HEK293E cells were starved from serum (full complement of culture ingredients except serum) and re-stimulated with 100 ng insulin for 10 min, and analyzed for P-S6K1(T389).
(D) Endogenous Rab1A was immunoprecipitated and analyzed for the presence of mTOR and Raptor.
(E) WT and mutant HA-Rab1A was transiently expressed in HEK293E cells. mTOR was immunoprecipitated and analyzed for its interaction with HA-tagged proteins and endogenous raptor. HA-Rap2A was a negative control.
(F) 32P-labeled HEK293E cells were starved and re-stimulated with AA. Rab1A was immunoprecipitated and analyzed for the bound 32P-labeled GTP and GDP by thin layer chromatography. Numbers at the bottom show means ± SD in three independent experiments.
(G) HEK293E cells were starved and re-stimulated with AA. Extracts of cells were incubated with GTP-Agarose beads and the binding of Rab1A was analyzed by Western blot. Bottom panel shows means ± SD in three independent experiments.
(H) HEK293E cells expressing Myc-mTOR and WT or mutant HA-Rab1A proteins were starved and re-stimulated with AA. Myc-mTOR and HA-Rab1A were analyzed for interaction by co-IP. HA-Rap2A is a negative control.
(I) Same as Figure 2H except mTORC1 signaling was analyzed by P-S6K1. Rheb is used as a positive control.
(J) Same as in Figure 2G except cells were starved and re-stimulated with serum. Bottom panel shows means ± SD in three independent experiments.
See also Figure S2