Src kinase-dependent Akt phosphorylation induced by combined G_12/13 and G_i/G_z signaling in platelets. (A) Gα_q-deficient platelets preincubated with either 10 μM Y-27632 or 10 μM PP2 were stimulated at 37°C for 2 minutes with either AYPGKF (500 μM) or thrombin (0.5 unit/mL). Platelet proteins were separated by SDS-PAGE, Western blotted, and probed for phospho-Tyr⁴¹⁶ Src. (B) Gα_q-deficient platelets preincubated in the absence and presence of 10 μM PP2 were stimulated at 37°C for 5 minutes with agonists as noted. The reaction was stopped by the addition of 3 × SDS sample buffer. Platelet proteins were analyzed by Western blot analysis with anti-Ser(P)⁴⁷³ antibody. (C) Washed human platelets preincubated with 1 μM AR-C69931MX, 10 μM PP2, or 10 μM PP3 were stimulated at 37°C for 5 minutes with either 2-MeSADP (1 μM) or epinephrine (10 μM). Platelets stimulated with 2-MeSADP were preincubated with 10 μM MRS-2179. Platelet proteins were separated by SDS-PAGE, Western blotted, and probed with anti-phospho-Akt (Ser⁴⁷³) antibody. The Western blot analysis shown is a representative of 3 independent experiments.