Figure 3.

Effects of E2β and S-nitrosylation on CFL1 phosphorylation. A, Subconfluent HUVECs were treated with 10 nM of E2β for the indicated time periods. Total protein extracts were harvested and analyzed by immunoblotting with antibodies against p^Ser3-CFL1 and CFL1. Representative blots of p^Ser3-CFL1 and total CFL1 of a typical experiment are shown. Graphs summarize data (mean ± SEM) from three independent experiments using cells from different fetuses. Data were normalized and expressed fold to control. *, P < .05 vs untreated control. B, HUVECs were pretreated with or without NOS inhibitor, L-NAME, and transfected with or without scrambled or eNOS-specific siRNAs, followed by treatment with or without E2β for 30 minutes. Representative blots of p^Ser3-CFL1, total CFL1, and eNOS of a typical experiment are shown. C, Flag-tagged CFL1 and the mutants were overexpressed in HUVECs, respectively. Cellular proteins were harvested for immunoblotting with antibodies against p^Ser3-CFL1 and CFL1. Total β-actin was used as a loading control. Representative blots of p^Ser3-CFL1, p^Ser3-Cfl1^flag, and total CFL1 of a typical experiment are shown. Data (mean ± SEM) were summarized in the lower graph from three independent experiments using cells from different fetuses. Bars with different letters differ from each other significantly (P < .05); paired comparisons of importance were indicated with an asterisk (P < .05).