IGF-1 stimulates phosphorylation of PKB in a PI3K-dependent manner. Washed platelets were stimulated with the indicated concentration of IGF-1 for 2 minutes (A), stimulated for the indicated times with IGF-1 (100 nM) (B), stimulated with IGF-1 (100 nM for 2 minutes), TPO (100 ng/mL for 10 minutes), leptin (100 ng/mL for 5 minutes), and SFLLRN (5 μM for 5 minutes) (C), or incubated with wortmannin (100 nM for 10 minutes), LY294002 (20 μM for 10 minutes), UO126 (10 μM for 10 minutes), bisindolylmaleimide I (BIM1; 5 μM for 10 minutes), rapamycin (200 nM for 10 minutes), ARC-69331-MX/A3P5P (1 and 100 μM for 3 minutes) and apyrase (2 U/mL for 3 minutes) prior to stimulation with IGF-1 (100 nM) for 2 minutes (D). Platelets were extracted and whole-cell lysate (WCL) was subjected to SDS-PAGE followed by immunoblotting with the anti-P–PKBSer473 antibody. The bar graph (B) represents quantification of the phosphorylation of PKB (ratio of phosphorylated/total) expressed as a percentage of the IGF-1 response at 2 minutes (means ± SEM; n = 7). Membranes were stripped and reprobed with anti-PKBα antibody to confirm equal loading. Results (A,C,D) are representative of 3 similar experiments.