FIG. 7.

GTP-Ras in 293T cells expressing wild-type GRF1 and L263Q mutant. Cells were transiently transfected with the indicated GRF1 gene. Cultures were serum starved and metabolically labeled with [³²P]orthophopshate, cells were treated with 5 μM ionomycin or 20 nM EGF for 5 min, extracts were immunoprecipitated with a Ras-specific monoclonal antibody, and the percentage of GTP-Ras was determined by thin-layer chromatography as described in Materials and Methods. Results in panel A represent the average of two experiments, while the results in panel B are from a single experiment.