IGF-1 stimulates tyrosine phosphorylation of the IGF receptor and downstream signaling to IRS-1, IRS-2, and p85 PI3K. Washed platelets were stimulated with the indicated concentration of IGF-1 for 2 minutes (A,F) or stimulated for the indicated times with IGF-1 (100 nM) (B-E). Platelets were extracted, and the IGF receptor (IGFR) (A,B), IRS-1 (C), IRS-2 (D), and p85 (E) were subsequently immunoprecipitated (IP). The immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies (PTyr, antiphosphotyrosine). The bar graph represents quantification of the phosphorylation of the IGF receptor (ratio of phosphorylated/total) expressed as a percentage of the IGF-1 response at 200 nM (A) or at 60 minutes (B) (means ± SEM; n = 3). Membranes were stripped and reprobed with anti–IRS-1 (C), anti–IRS-2 (D), and p85 (E) to confirm equal loading. Results are representative of 3 similar experiments.