Fig. 2
Effects of S1PC on TNF-α-induced phosphorylation and interaction with Rho family of GEF-H1 in HUVECs. a Effect of S1PC on TNF-α-induced GEF-H1 phosphorylation in HUVECs. Cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 0.25, 0.5, 1 or 3 h and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group with the same treatment time (*p < 0.05) was determined by Bonferroni’s multiple comparison test. b The concentration-dependent effect of S1PC on TNF-α-induced GEF-H1 phosphorylation in HUVECs. Cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (75, 150 or 300 μM) for 30 min and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 4. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (**p < 0.01) was determined by Dunnett’s multiple comparison test. c Effect of S1PC on TNF-α-induced interactions of GEF-H1 with Rho family proteins, RhoA (right graph) and Rac (left graph) in HUVECs. Cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 1 h and immunoprecipitated with anti-GEF-H1 antibody (IP: GEF-H1) or control antibody (IgG). Then the precipitated proteins and total proteins (Input) were analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3–4. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (**p < 0.01) was determined by Bonferroni’s multiple comparison test