Figure 6.

c-Src activates Vav2 to regulate Nox1-dependent ROS formation. (A) Inhibition of Src activity in HT29 cells by PP2 treatment reduces ROS generation and blocks endogenous Vav2 tyrosine-phosphorylation. HT29 cells were treated with 10 μM Src inhibitor PP2 or with its nonfunctional analogue PP3, and ROS production was monitored (left). One representative experiment from three separate experiments is shown, and results are given as mean of triplicates ± SD. The inhibition of Src activity by PP2 blocks tyrosine-phosphorylation of endogenous Vav2 without affecting the expression level of endogenous c-Src and Vav2 (right). The results shown are typical of at least three separate experiments. (B) Vav2-specific siRNAs that strongly decrease endogenous Vav2 protein levels also potently decrease ROS generation in HT29 cells. HT29 cells were transfected as described in Materials and Methods with three different Vav2-specific siRNAs (20 nM), and after 72 h the ROS formation was quantified by CL-assay (left). The right panel shows that all three Vav2-specific siRNAs significantly decrease endogenous Vav2 protein levels, with oligo#2 being the most effective. One representative experiment from three separate experiments is shown, and results are given as mean of triplicates ± SD. (C) SrcYF-induced ROS generation in HT29 cells is blocked by the transfection of Vav2-specific siRNA#2, which strongly decreases the level of endogenous Vav2 protein. HT29 cells were transfected by RNAiMax with empty vector or SrcYF (as indicated), and with control siRNA or with Vav2 siRNA#2. After 48 h, ROS generation was measured by CL-assay. One representative experiment from three separate experiments is shown, and results are given as mean of triplicates ± SD.