Fig. 3.

Effect of chronic down-regulation of the mTORC1/S6K1 pathway on the activation of the IRS-1/PI3K pathway in 3T3-L1 adipocytes. After siRNA or rapamycin treatment, cells were serum deprived and stimulated for 45 min with 10 nm insulin before lysis and subsequent analysis of IRS-1 phosphorylation and PI3K activity. Western blotting of cell lysates (A and B) or immunoprecipitated IRS-1 (C) with antibodies against (A) IRS-1 Ser636/639 (n = 5), (B) IRS-1 Ser1101 (n = 3), and (C) phosphotyrosine (n = 3). D, IRS-1 associated PI3K activity (n = 8). Densitometry measures of phosphorylation and kinase activity were normalized for the expression of IRS-1 and for protein content, respectively. Results show representative gels and densitometric analysis. Results are shown as means ± se. Groups with different letters are significantly different (P < 0.05).