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.