Fig. 5. Enhancement of actin dorsal ruffle formation by 480-Cbl requires Src, PI 3-kinase and Rac. (A) Following serum starvation, 480-Cbl-expressing NIH 3T3 cells were treated for 20 minutes with either DMSO carrier, PKC inhibitor BIM I (5 µM), Rac inhibitor SCH 51344 (75 µM), Src inhibitors SU6656 (5 µM) or PP2 (5 µM), PI 3-kinase inhibitors Wortmannin (0.5 µM) or LY294002 (30 µM), or left untreated, as indicated. Cells were fixed and stained with TRITC phalloidin to quantify actin dorsal ruffle formation after 5 minutes of activation by FCS (10%), LPA (2.5 µM) or PDGF (10 ng/ml) as indicated. (B) 480-Cbl expressing cells were transiently transfected with NLS-GFP (panel 1), NLS-GFP and R296/F528-Src (panel 2), NLS-GFP and N17-Rac1 (panel 3) and NLS-GFP and p85iSH2N (panel 4). The cells were treated with PDGF for 5 minutes following serum starvation and the NLS-GFP and TRITC phalloidin staining of representative samples were visualized by confocal fluorescence microscopy. The number of NLS-GFP-positive cells forming distinct actin dorsal ruffles is indicated in each panel. Bar, 30 µm.