Total and phosphorylated Akt in WT and Akt3^−/− platelets. (A,D) 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 directed against: (A) Akt3, total Akt (Akt1, Akt2, and Akt3), phosphorylated Ser473 of Akt, and GSK-3β (loading control), and (D) phosphorylated Thr³⁰⁸ of Akt, total Akt and α-tubulin (loading control). (B,C) Western blot results from each of 3 experiments as shown in panel A were scanned and quantitated using NIH ImageJ for uncalibrated optical density. The relative quantity of total Akt (B) and phosphorylated Ser473 of Akt (C) in wild-type and Akt3^−/− platelets are shown (mean ± SE). (E) Washed Akt3^−/− and WT mouse platelets were solubilized and immunoblotted with antibodies directed against Akt1, Akt2, and Akt3 and α-tubulin (loading control).