Figure 4.

c-Src activity regulates the levels of Rac1-GTP in HT29 cells. (A) The inhibition of Src activity significantly lowers the levels of active Rac1 in HT29 cells. HT29 cells were treated Src inhibitor PP2, its nonfunctional analogue PP3, or DMSO alone. Total Rac and Rac1-GTP levels were measured by PBD assay as indicated in Materials and Methods. A nonhydrolysable analogue of GTP, GTPγS, was used as positive loading control and GDP was used for the negative control. Inhibition of Src activity was confirmed by Western blotting of the Src tyrosine phosphorylation target pTyr418-Vav, whereas similar levels of total Vav protein in HT29 cells was confirmed by Vav immunoblot. Finally, equal loading of lanes was confirmed by actin immunoblot. (B) PP2 treatment significantly lowers the levels of active Rac1 in HT29 cells compared with treatment with PP3 or DMSO alone. The results from three independent experiments, performed as described above, were quantified, normalized to the value of DMSO-treated cells, and they are shown as mean ± SD.