Fig. 4

Effect of S1PC on a downstream signaling of TNF-α, Rac, in HUVECs. a Effect of S1PC on TNF-α-induced Rac activity in HUVECs. The active GTP-bound Rac (GTP-Rac) was obtained with Rac activation assay system after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 1 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 < 0.05) or TNF-α-treated group (*p < 0.05) was determined by Bonferroni’s comparison test. b Effects of S1PC on TNF-α-induced phosphorylation of ERK1/2 (upper graph) and Elk1 (lower 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 5, 15, 30 or 60 min 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, ^#p < 0.05) or TNF-α-treated group with the same treatment time (*p < 0.05) was determined by Bonferroni’s multiple comparison test. c, d Effects of S1PC on TNF-α-induced gene (c) and protein (d) expression of MLCK in HUVECs. Total mRNA or cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 9 or 24 h and analyzed by quantitative real-time PCR analysis or western blotting with indicated antibodies, respectively. 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, *p < 0.05) was determined by Bonferroni’s comparison test