Fig. 3. Src kinase inhibitor PP1 inhibits Shc but not IRS-1 tyrosine phosphorylation by IGF-I. A, proliferating and growth-arrested 3T3-L1 cells were treated with or without 10 µM PP1 for 30 min prior to stimulation with 10 nM IGF-I for 5 min. The immunoprecipitates (IP) were resolved by SDS-PAGE and transferred to membranes. Top two panels, Western blots of Shc IPs exposed first to phosphotyrosine antibodies, then stripped and exposed to Shc antibodies. Bottom three panels, Western blots of IRS-1 IPs exposed first to phosphotyrosine, then p85, and finally IRS-1 antibodies. B, IGF-I-stimulated phosphotyrosine (PY) of Shc and IRS-1, compiled from three separate experiments and corrected for total p52 Shc and total IRS-1, was analyzed by densitometry of Western blots and expressed as fold increase (mean + SE) in IGF-I-stimulated PY from control cells (open bars) or PP1-treated cells (solid bars). The differences in the PP1-treated and untreated groups were analyzed using ANOVA with a Tukey-Kramer post hoc test. *, significance at P < 0.001.