Fig. 2.

Role of Phospho-Ser-302 in the Subsequent Ser-318 Phosphorylation

A, C2C12 cells transfected with empty vector (con), IRS-1 wt, or IRS-1 A_302/307 were stimulated with 10 nm insulin as indicated. A representative immunoblot for phospho-Ser-318 and the reprobe for IRS-1 are shown. In the lower part, densitometric quantification of the immunoblots is shown (n = 4; mean ± sem); *, P < 0.05 compared with IRS-1 wt transfected cells stimulated with insulin for 5 min. (B and C) C2C12 cells transfected with IRS-1 wt or IRS-1 E₃₀₂A₃₀₇A₃₁₈ (B) and IRS-1 wt or IRS-1 A₃₀₂ (C) were stimulated with 10 nm insulin for 5 min. PKC-ζ/λ was immunoprecipitated and coprecipitated IRS-1 was visualized by immunoblotting. IRS-1 protein expression in the extracts is shown in the upper panel. The lower part of the figure shows the densitometric quantification (n = 5; mean ± sem); *, P < 0.05 vs. IRS-1 wt transfected cells stimulated with insulin for 5 min; #, P < 0.05 vs. unstimulated IRS-1 wt transfected cells. con, Control; IB, immunoblot; ins, insulin; IP, immunoprecipitation; rel., relative.