Fig. 3. Effect of the Grb2-SH2 blocking peptide on growth factor-mediated ERK activation in vivo. A, NIH-3T3 cells. The Grb2-SH2 blocking peptide was electroporated into NIH-3T3 cells growing on fully conductive slides (Fig. 1 B) and growth-arrested by serum starvation. After a 5-min incubation in DMEM, cells were stimulated with 100 ng/ml EGF (Lanes 24) for 5 min. Proteins in detergent cell lysates were resolved by PAGE and analyzed by Western blotting using the antibody against the dually phosphorylated active ERK enzymes (see "Materials and Methods"). Lane 1, control unstimulated cells. Lane 2, control nonelectroporated EGF-treated cells. Lane 3, cells electroporated with the control phenylalanine-containing peptide and EGF-stimulated. Lane 4, cells electroporated with the Grb2-SH2 binding peptide and EGF-stimulated. B, methu-expressing NIH-3T3 cells. The Grb2-SH2 binding peptide containing Pmp at the position of phosphotyrosine (Lane 4), a control phenylalanine-containing peptide (Lane 3), or the EGFR phosphorylation inhibitor, compound 8 (Lanes 10 and 12), was electroporated into NIH-3T3-methu cells. Cultures were subsequently stimulated with HGF (Lanes 25, 7, and 8), EGF (Lanes 10 and 11), or PDGF (Lanes 12 and 13), as indicated. Lanes 1, 6, and 9, unstimulated controls. Lane 5, cells treated with 100 µM of the MEK inhibitor PD98059 for 2 h and stimulated with HGF. Lane 7, cells treated with 40 µM of the PI3k inhibitor LY294002 for 2 h and stimulated with HGF. After treatment, cell extracts were probed for activated ERK as described above. Arrows point to the position of the p42 (ERK2) and p44 (ERK1) forms of the activated ERK kinases.