(A–C) Immunoblot analysis of phosphorylation of AKT after growth factor treatment in HEK293T and PC12 cells over-expressing Rhes. Cells were serum-starved for 20 h, and treated with 5% serum (A), 50 ng/ml IGF-1 (B), other growth factors (25 ng/ml EGF, 50ng/ml PDGF, or 100ng/ml FGF) for indicated time point (C).
(D) Inducible T-REx HEK293 stable cell lines that express Myc or Myc-Rhes were either left untreated or induced by 1μg/ml tetracycline. After 6 h, cells were incubated serum-free media in the absence or presence of tetracycline for 16h followed by 25ng/ml IGF-1 treatment for 10 min. Cell lysates were analyzed for P-AKT.
(E) Differentiated rat phaeochromocytoma (PC12) cells that express Myc or Myc-Rhes were serum-starved for 20 h, and treated with 25ng/ml EGF for 5 min.