Figure 4.

Effects of chronic ET-1 treatment on insulin-stimulated tyrosine phosphorylation of Gαq/11 and its association with p110α. After pretreatment with 25 μM LY294002 (LY), 20 nM Rapamycin (Rapa), or 0.1% DMSO vehicle for 30 minutes, 3T3-L1 adipocytes were incubated with or without ET-1 (10 nM) for 24 hours and then stimulated with insulin (100 ng/ml) for 0–10 minutes. Cells were lysed and immunoprecipitated with anti-Gαq/11 antibody. Immunoprecipitates were analyzed by Western blotting using PY-20 antibody (a and c, both upper panels), anti-p110α antibody (b, upper panel, and c, middle panel), or anti-Gαq/11 antibody (c, bottom panel). Whole cell lysates were analyzed by Western blotting using anti-Gαq/11 antibody (a and b, both lower panels) as described in Methods. These experiments were repeated twice.