Figure 6.

Effect of echistatin on tyrosine phosphorylation of IRS-2 after IGF-I stimulation. Subconfluent SMCs were incubated without echistatin (lanes 1 and 2) or with echistatin at 10⁻⁷ M (lane 3) for 12 h in serum-free DMEM-H. The cells were exposed to no IGF-I (lane 1) or 100 ng/ml (lanes 2 and 3) for 10 min. They were then lysed with 1 ml of RIPA buffer, and the lysates were immunoprecipitated by using an anti-IRS-2 antiserum as described in Materials and Methods. The precipitated proteins were separated by 7.5% SDS/PAGE and transfered to a PVDF membrane. The membrane was blotted with anti-phosphotyrosine antibody (A) or an antibody against IRS-2 (B).