Phosphorylation of GSK-3β. (A) Washed Akt3^−/− and WT mouse platelets were stimulated with thrombin (0.018 U/mL) for 1, 3, and 5 minutes, solubilized, and immunoblotted with antibodies against phosphorylated Ser9 of GSK-3β, and total GSK-3β (loading control). (B) Western blot results from each of 3 experiments as shown in panel A were scanned and quantified using NIH Image J for uncalibrated optical density. The relative quantity of phospho-GSK-3β in thrombin stimulated Akt3^−/− versus that of wild-type mouse platelets is expressed as the percentage of wild-type (mean ± SE, 5 experiments). The difference in GSK-3β phosphorylation at resting, 1 minute, 3 minutes, and 5 minutes time points are significant between WT and Akt3^−/− (P < .05), as determined using Student t test. (C) Washed WT mouse platelets were preincubated for 2 minutes with increasing doses of SH-6 or vehicle control DMSO and stimulated with thrombin (0.018 U/mL) for 3 minutes, solubilized, and immunoblotted with antibodies against phospho-Ser9 of GSK-3β, phospho-Ser473 of Akt, total Akt, and total GSK-3β (loading control).