Fig. 4. Specificity of the inhibitory activity of the Grb2-SH2 blocking peptide. The Grb2-SH2 blocking peptide (A, B, E, F, G, and H) or the control phenylalanine-containing peptide (C and D) was introduced by in situ electroporation into NIH-3T3 (A–D), methu-expressing NIH-3T3 (E and F) or A549 (G and H) cells growing on partly conductive slides (Fig. 1 A) and growth-arrested in spent medium. Five min after electroporation, cells were stimulated with EGF for 5 min (A–D) or HGF for 20 min (E–H), fixed, probed for activated ERK (see "Materials and Methods"), and photographed under bright-field (A, C, E, and G) illumination. B, D, F, and H, same frames as in A, C, E, and G, respectively, phase-contrast illumination. Magnification: A and B, x240; C–F, x40. The arrow points to the transition line between the stripped (b) and electroporated (a) areas, whereas the arrowhead points to the line between the control ITO-coated (c) and etched (b) areas (Fig. 1 A). Cells growing on the left side (a) were electroporated, whereas cells on the stripped zone (b) or right side (c) of the slide did not receive any pulse. Note that the Grb2-SH2 blocking peptide dramatically reduced the EGF signal (A, a), whereas the degree of ERK activation is the same on both sides of the slide (a or c) for cells electroporated with the control phenylalanine-containing peptide (C). In A, the inhibition of the signal extends into ~3–4 rows of adjacent cells in the nonelectroporated area (b), probably due to movement of the peptide through gap junctions (27) . On the other hand, the Grb2-SH2 blocking peptide caused only a slight reduction of the HGF signal (E and G, area a). At the same time, there is no detectable effect on cell morphology as shown by phase-contrast microscopy (B, D, F, and H).