|
Fig. 7 Sema3F inhibits VEGF-induced phosphorylation of Akt and eNOS. (A) Sema3F inhibits VEGF-mediated phosphorylation of Akt at Ser473. HUVECs were treated with VEGF (50 ng/ml) and Sema3F (10 ng/ml or 100 ng/ml) or solvent for 30 min. HUVECs were lysed and proteins were extracted to perform Western blot analysis of Akt, pAkt (Ser473) and α-tubulin. α-tubulin served as the loading control. Bands were quantified by densitometric analysis. Representative blots from four independent experiments are shown (n, number of biological replicates). (B) SiRNA-mediated Sema3F knockdown increased phosphorylation of Akt at baseline. HUVECs were transfected with Sema3F siRNA or scrambled siRNA (scrsiRNA). 24 h post transfection cells were treated with VEGF (50 ng/ml) or solvent for 15 min. Cell lysates were used for Western blotting. Representative blots from five independent experiments are shown (n, number of biological replicates). (C) Sema3F inhibits VEGF-induced phosphorylation of eNOS at Ser1177. HUVECs were incubated with VEGF (50 ng/ml), Sema3F (100 ng/ml) or both for 15 min and then lysed. Phosphorylation of eNOS (ser1177) was analyzed by Western blotting with an anti-phospho-eNOS antibody. α-tubulin served as loading control. Representative blots from four independent experiments are shown (n, number of biological replicates). (D) SiRNA-mediated Sema3F knockdown enhanced phosphorylation of eNOS at baseline. HUVECs were transfected with siSema3F or scrsiRNA. 24 h later the cells were stimulated with or without VEGF (50 ng/ml) for 15 min. Representative data from four independent experiments are shown (n, number of biological replicates). (A-D) Data were analyzed by the one-way ANOVA test followed by Bonferroni's multiple comparisons test. *p < 0.05. All box plots extend from the 25th to the 75th percentile with the median marked. Whiskers extend from the 10th to the 90th percentile.