FIGURE 1.

Temporal phosphorylation of different Akt isoforms, GSK3, PRAS40, and pleckstrin in human platelets. A–D, washed human platelets were stimulated with 0.2 unit/ml thrombin (A), 5 μm SFLLRN (B), or 100 μm AYPGKF (C) for the indicated time period before extraction in NuPAGE sample buffer and immune blotting with the indicated antibodies (A–C). In addition, mouse brain lysate (positive control) and mouse and human platelet (SW, single washed; DW, double washed) lysates were immunoblotted for Akt3 using two different antibodies (mAb62A8 (i) and pAb (ii) (D). E–G, alternatively, platelets were stimulated with 5 μm SFLLRN for the indicated time period and extracted in Nonidet P-40 extraction buffer followed by immunoprecipitation (IP) of Akt1 (E), Akt2 (F), and Akt3 (G). Akt phosphorylation was analyzed by immunoblotting with the indicated antibodies (i), and in vitro Akt activity was analyzed using the peptide substrate RPRAATF (ii). Bar graphs (ii) show activity expressed as pmol of P_i incorporated per minute for 10⁸ platelets (mean ± S.E. (error bars), n = 3). H, human (i) and mouse (ii) platelets were stimulated with thrombin (0.2 unit/ml), AYPGKF (100 nm), and SFLLRN (5 μm) for 5 min and extracted in Nonidet P-40 followed by immunoprecipitation of Akt3. Akt3 immunoprecipitates (Akt3 IP) and whole cell lysates (WCL) were immunoblotting with the indicated antibodies. Results (A–H) are representative of at least three independent experiments.