Fig. 8. Src and PI 3-kinase mediate the increased Rac-activation by 480-Cbl expression. (A) Following serum starvation, 480-Cbl-expressing NIH 3T3 cells were treated for 20 minutes with either DMSO carrier, PKC inhibitor BIM I (3 µM), Src inhibitors SU6656 (5 µM) or PP2 (10 µM) or PI 3-kinase inhibitor LY294002 (30 µM), as indicated. Cells were lysed either after no further treatment or after 1 minute of activation by PDGF (10 ng/ml). Aliquots of the lysates were either directly subjected to SDS-PAGE and probed by western blotting with anti-Rac antibodies or were incubated with sepharose-bound GST-PAK-CD and bound proteins subjected to SDS-PAGE and western blotting with anti-Rac antibodies. (B) Serum-starved NIH 3T3 expressing either full-length c-Cbl or truncations at amino acids 655, 563, 528, 480 (with and without the TKB domain inactivating G306E mutation) and 388 were treated with PDGF for 5 minutes. Microfilaments were visualized by confocal fluorescence microscopy following Tritc-phalloidin staining, and the percentage of cells that form actin dorsal ruffles was quantified (see Materials and Methods).