Fig. 4. Dominant-negative SrcRF mutant inhibits IGF-I-stimulated MAPK activation. A, proliferating 3T3-L1 cells were mock-transfected (control) or transfected with wild-type c-Src (WT c-src) or SrcRF mutant (srcRF) at a transfection efficiency of ~70%. Twenty-four h after transfection, cells were incubated overnight in SFM and then stimulated with 10 nM IGF-I for 5 min. Cell lysates were analyzed for MAPK activation by Western blotting for phospho-MAPK (A) and then total MAPK (not shown). B, phospho-MAPK unstimulated (open bars) and after IGF-I stimulation (solid bars) from two separate transfection experiments (n = 4, each condition) was normalized for the total amount of MAPK and expressed as densitometric units (mean + SE).