FIGURE 5.

α-Syn overexpression inhibits small GTPase RalA activity in HUVECs. After transfection of HUVECs with GFP-α-syn (A) and GFP-α-syn deletion mutants (B), the cells were treated with 100 nm PMA, 2 units/ml thrombin, or 20 μm forskolin together with 100 μm IBMX for 1 h. GTP-bound form of RalA was then isolated from cell lysates incubated with the GST-RalBP and analyzed by Western blot with anti-RalA monoclonal antibody. The data were analyzed by the band intensities of Western blot. C, after cotransfection of HUVECs with α-syn and active RalA mutant (Q72L) and treatment with 100 nm PMA for 1 h, the expression level of α-syn and RalA was detected by Western blot. con, control. D, after stimulation with 100 nm PMA for 1 h, the cell-free supernatants were then analyzed for vWF by ELISA. *, p < 0.05; **, p < 0.01.