Figure 4. Gab1 is important for the activation of mTORC signaling after EGF stimulation.
(A) T24 cells were transfected with Gab1 siRNA, treated with LY294002 (20 μM) for 60 min, stimulated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473, ERK-pT202/Y204 and Gab1. (B) E6 cells transfected with Myc-Gab1wt were serum-starved, and the treatment was performed in a similar manner to that described in A. Immunoblotting was performed to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473, ERK-pT202/Y204, Gab1 and myc. (C) E6 cells were transfected with Myc-Gab1wt and Myc-Gab1ΔPH, treated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473 and Gab1. (D) T24 cells were transfected with raptor siRNA, rictor siRNA and Myc-Gab1wt, treated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, raptor, rictor, p70S6K-pT389, AKT-pS473, Gab1 and myc. For all experiments, n = 3.