FIGURE 2.

Plasmin-induced serine phosphorylation of A2 is specifically mediated by classical PKC. A, CMECs were treated with either plasmin (0.3 unit/ml, 15 min; left), trypsin (2.76 units/ml, 3 min; middle), or thrombin (2 units/ml, 7 min; right). Total serine- or tyrosine-phosphorylated proteins (1000, 1000, and 550 μg, respectively) were immunoprecipitated with anti-phosphoprotein IgGs and then immunoblotted with monoclonal anti-A2 IgG. Total A2 and phospho-PKC were also evaluated by immunoblotting. B, HUVECs were transfected with siRNA directed against PKCα (1 μm) or control siRNA for 72 h, washed, and then treated with plasmin for the indicated time periods. Expression of phospho-PKC, serine phospho-A2, and phospho-ERK1/2 was monitored by immunoblotting. Specificity of the knockdown was determined by immunoblot analysis of total PKCα and PKCδ protein.