Figure 6.

Effects of chronic ET-1 treatment on insulin-stimulated tyrosine phosphorylation of IRS-1, IRS-2, Gab-1, Cbl, and insulin receptor β-subunit. Serum-starved 3T3-L1 adipocytes were incubated in the absence or presence of insulin (100 ng/ml) for 5 minutes, after treatment with or without ET-1 (10 nM) for 24 hours. (a) Whole-cell lysates were subjected to SDS-PAGE and immunoblotted with PY-20 antibody (a, top panel) or anti-insulin receptor (IR)-β antibody (a, lower panel). (b) Cell lysates were immunoprecipitated with anti-IRS-1 antibody and were analyzed by Western blotting using anti-p110α antibody (middle panel). Whole-cell lysates were subjected to SDS-PAGE and immunoblotted with PY-20 antibody (top panel) or anti–IRS-1 antibody (lower panel). (c) Cell lysates were immunoprecipitated with anti–IRS-2, Gab-1, or Cbl antibody, and were analyzed by Western blotting using PY-20 antibody (first, third, and fifth panels), and anti-IRS-2, Gab-1, or Cbl antibody (second, fourth, and sixth panels) as described in Methods. These experiments were repeated twice.