Figure 4.

Effect of different concentrations of PDGF-BB on tyrosine phosphorylation of PDGF-Rβ, p44^mapk/p42^mapk, and PI 3′-K. Confluent cells in 75-cm² flasks were preincubated in serum-free medium for 24 h. Then VSMCs were stimulated with 1–50 ng/ml PDGF-BB for 5 min. Then the cells were lysed, and tyrosine-phosphorylated proteins were immunoprecipitated using an anti-phosphotyrosine antibody coupled to Sepharose. Proteins (5 μg) were analyzed by 7.5% SDS-PAGE. Tyrosine-phosphorylated PDGF-Rβ, p44^mapk/p42^mapk, and PI 3′-K were detected by the enhanced chemiluminescence method using the respective monoclonal antibodies.