Fig 6: Gab2 silencing suppresses thrombin-induced signaling and inflammation in endothelial cells.

(A) HUVEC were transfected with 200 nM scrambled siRNA (scRNA), Gab1, or Gab2 siRNA. After 48 h, the cells were treated with thrombin (10 nM) for 6 h. The cell lysates were analyzed for VCAM1 and ICAM1 protein levels by immunoblot analysis. Band intensities were quantified by densitometry, and these data were shown in the right side panel. (B) HUVEC transfected with scrambled, Gab1, or Gab2 siRNA were treated with thrombin for 6 h and the MCP-1 levels were analyzed in the cell supernatants by ELISA. (C) HUVEC transfected with Gab1 or Gab2 siRNA were treated with thrombin for indicated time points and the activation of ERK, p38 MAPK, and NF-κB was analyzed by immunoblot analysis. Band intensities were quantified by densitometry, and these data were shown in the right side panel. (D) HUVEC were transfected with a scrambled siRNA or siRNA specific for c-Src, Fyn, or Yes. After 48 h, the cells were treated with thrombin for 5 min. Gab2 was immunoprecipitated and probed for tyrosine phosphorylation using the pY monoclonal antibody in immunoblot analysis. Band intensities were quantified by densitometry, and these data were shown in the right side panel. Data were representative of three independent experiments with similar results. One-way analysis of variance (ANOVA) was used to compare the data of experimental groups, Tukey’s post hoc multiple comparison test was used to obtain statistical significance. ***, p<0.001, **, p<0.01; *, p<0.05; ns, no statistically significant difference.