Figure 9.

Effects of chronic ET-1 treatment on insulin-stimulated MAPK and SHC phosphorylation. Serum-starved 3T3-L1 adipocytes were incubated in the absence or presence of 100 ng/ml insulin (I) or 50 ng/ml PDGF (P) for 5 minutes, after treatment with or without ET-1 (10 nM) for 24 hours. Cell lysates were immunoprecipitated with anti-SHC antibody and were analyzed by Western blotting using PY-20 antibody (a, top panel). In experiments, whole-cell lysates were analyzed by Western blotting using anti-SHC (a, bottom panel), phospho-specific MAP-kinase (P-MAPK) (b, top panel), or anti-ERK-1 antibodies (b, lower panel), as described in Methods. These experiments were repeated twice.