Akt phosphorylates PDE3A during thrombin activation of platelets. (A) Washed platelets were stimulated with thrombin at 37°C for 3 minutes. The samples were separated in 4%-15% SDS-PAGE gels and transferred into PVDF membranes. The membranes were probed by anti-Akt mAb, antiphospho-Akt (Ser473) mAb, or anti-phospho-Akt substrate mAb, respectively. (B) The purified PDE3A protein were immunodetected with anti-phospho-Akt substrate mAb. Lanes 1 and 2 represent the purified platelet PDE3A proteins from the vehicle control platelets and the thrombin-treated platelets, respectively (thrombin at 0.25 nmol/L). The data are representative of the 3 similar experiments. (C) Identification of thrombin-induced PDE3A phosphorylation with immunoprecipitated method. Washed platelets incubated with AktI (2 μmol/L) or vehicle were treated by thrombin (0.25 nmol/L at 37°C for 3 minutes) or vehicle. Platelet lysates were immunoprecipitated by the anti-Akt phosphorylated-substrate monoclonal antibody and immunoblotted by PDE3A antibody.