GSK3β is present and phosphorylated after agonist treatment in platelets. (A) Lysates of washed human or WT mouse platelets (10⁸) and mouse liver (20 μg/mL) were immunoblotted with an antibody to GSK3β. The second immunoblot shows a comparison of 10⁸ platelets from WT mice and GSK3β^+/− mice immunoblotted with an antibody to GSK3β. The blot was reprobed with an antibody to Akt. (B) Washed human and mouse platelets and HeLa cells (20 μg/mL) were immunoblotted with an antibody to GSK3α. (C) Washed human platelets were stimulated with ADP (10 μM) or thrombin (0.1 u/mL) for 10 minutes with or without ARL66096 (300 nM), A3P5PS (300 μM), or PP2 (50 μM). Lysates were then immunoblotted for Akt phospho-(ser473), GSK3β phospho-(ser9), and total Akt. The mean (± SD) of the fold increase in phosphorylation compared with untreated control for 3 experiments is shown in the graph above a representative experiment. (** indicates a significant difference compared with control with P ≤ .01 according to an unpaired Student t test).