Figure 8.

Effects of Q209L-Gαq on IRS-1 protein and insulin-stimulated tyrosine phosphorylation of IRS-1. (a) Differentiated 3T3-L1 adipocytes were infected with adenoviruses overexpressing either wild-type Gαq protein (WT-Gαq), constitutively active Gαq mutant (Q209L-Gαq), or mock control in 3T3-L1 adipocytes. After 60 hours of infection, cells were pretreated with 25 μM LY294002 (LY), 20 nM Rapamycin (Rapa), or 0.1% DMSO vehicle for 1 hour, incubated with or without insulin for 5 minutes as indicated, lysed, and immunoprecipitated with anti–PY-20 antibody. Immunoprecipitates were analyzed by Western blotting using anti-IRS-1 antibody (a, top panel). Whole-cell lysates were analyzed by Western blotting using anti-IRS-1 (a, middle panel, and b), or anti–insulin receptor β-subunit (a, bottom panel) antibodies, respectively. These experiments were repeated twice.