Figure 4

Src family kinase mediates TβRII-dependent TGFβ activation of RhoA and ATF2 in VSMCs. (A) Src family kinase inhibitor SU6656 dose-dependently reduces TGFβ activation of ATF2 but not that of Smad2. VSMCs were pretreated with increasing concentrations of SU6656 for 30 min and then stimulated with TGFβ for 10 min. Phosphorylation of ATF2 and Smad2 was determined by Western blot analysis. The membranes were subsequently probed with actin for loading control. ^#P < 0.05 vs. TGFβ-stimulated but without SU6656 treatment group. (B) SU6656 abolishes TGFβ-induced RhoA activation. VSMCs were pretreated with 5 μM SU6656 for 30 min prior to stimulation with TGFβ for 10 min. RhoA activation was then determined by GST-Rhotekin-RBD assays. GTP-RhoA was subsequently eluted and subjected to Western blot analysis with an anti-RhoA antibody. To verify equal loading, 15 μg of cell lysates were run on separate gels and blots probed with an anti-RhoA antibody for total RhoA. *P < 0.05 vs. control (− TGFβ and – SU6656); ^#P < 0.05 vs. TGFβ-stimulated vehicle group. (C) RhoA knockdown reduces TGFβ activation of ATF2 but not Smad2. VSMCs were transfected with 20 nM control siRNA or RhoA siRNA and then stimulated with or without TGFβ for 10 min. Western blot analysis was performed to detect RhoA and phosphorylation of ATF2 and Smad2. ^#P < 0.05 vs. TGFβ-stimulated siControl group. (D) RhoA knockdown reduces TGFβ-induced CRP2 expression. VSMCs were transfected with 20 nM control siRNA or RhoA siRNA and then stimulated with or without TGFβ for 24 h. Western analysis was performed to detect RhoA and CRP2 protein levels. The membranes were subsequently probed with actin for loading control. ^#P < 0.05 vs. TGFβ-stimulated siControl group. Representative blots of at least three independent experiments are shown.