FIG. 8.

(A) Effect of IRS protein overexpression on MAP kinase phosphorylation in embryonic fibroblast cell lines. After overnight serum starvation, cells were stimulated with 10 nM IGF-1 for 10 min at 37°C. Cell lysates were subjected to SDS-PAGE and analyzed by Western blotting with anti-phospho-p44/42 MAP kinase antibody (Tyr204). Blots were visualized by autoradiography (upper panel) and quantified by scanning densitometry (lower panel). Quantitative results are expressed as percentages of the level present in IGF-1-induced WT cells and are presented as the mean and SEM of three independent experiments. (B) Association of IRS proteins with Grb-2. Serum-starved cells were stimulated with 10 nM IGF-1 for 3 min at 37°C. Protein samples were immunoprecipitated (IP) with anti-Grb-2 antibody and analyzed by immunoblotting with anti-PY (upper panel) or anti-Grb-2 antibody (lower panel). Arrows indicate the migration of proteins immunoreactive with each IRS protein-specific antibody. (C) Grb-2 binding with PY proteins. Serum-starved cells were stimulated with 10 nM IGF-1 for 3 min at 37°C. Protein samples were immunoprecipitated with anti-PY antibody and analyzed by immunoblotting with anti-Grb-2 antibody.