FIGURE 3.

Grb7-mediated Ras activation is essential for ERK phosphorylation. A, cell lysates from the shLuc- or shGrb7-infected (shGrb7-12 or shGrb7-13) Sk-Br3 cells were subjected to GST-Raf-RBD pulldown assays and/or Western blotting with various antibodies, as indicated. There is undetectable endogenous Grb7 in NIH3T3 cells that was used as a control. p indicates phosphorylation. B, cell lysates from NIH3T3 cells transfected with either pKH3-Grb7 or vector alone and then subjected to a GST-Raf-RBD pulldown followed by Western analysis. For all experiments, cells were serum-starved for 24 h and stimulated by 10 ng/ml EGF for 15 min prior to collection of the cell lysates. IP, immunoprecipitation; WCL, whole cell lysate. p indicates phosphorylation.