Fig. 5. Quantitation of ERK activation by densitometry of immunocytochemically stained cells. NIH-3T3 (A), methu-expressing NIH-3T3 (B and C), or A549 (D) cells were plated on partly conductive slides (Fig. 1 A) and electroporated with the control phenylalanine-containing peptide or the Grb2-SH2 binding peptide, as indicated. After stimulation with the indicated growth factors (100 ng/ml TPA for 2, 10, or 30 min or inactive TPA analogue 4-phorbol-12,13-didecanoate for 10 min), cells were probed for activated ERK as described in the Fig. 4 legend. Densitometry was subsequently carried out using an MCID M5 image analysis program. Values represent staining intensity compared with the nonelectroporated side, with the average intensity of EGF-stimulated NIH-3T3 cells taken as 100%. Results are representative of at least three experiments. Error within each group is less than 10%. phe, control peptide containing phenylalanine at the position of the EGFR-Tyr1068. Pmp, Grb2-SH2 blocking peptide containing Pmp at the position of the EGFR-Tyr1068 4PDD, 4-phorbol-12, 13-didecanoate.