Role of G_12/13 signaling in potentiation of Akt phosphorylation in Gα_q^-/- mice. (A) Akt phosphorylation in response to AYPGKF and thrombin in Gα_q^-/- mice. Wild-type platelets and Gα_q-deficient platelets preincubated in the absence and presence of 1 μM AR-C69931MX were stimulated at 37°C for 5 minutes with either AYPGKF (500 μM) or thrombin (0.5 unit/mL). The reaction was stopped by the addition of 3 × SDS sample buffer. Equal amounts of proteins were analyzed by Western blot analysis with anti-Thr(P)³⁰⁸ or anti-Ser(P)⁴⁷³. The data shown are representative of 3 experiments. (B) G_12/13 signaling potentiates Akt phosphorylation in Gα_q-deficient platelets. Gα_q-deficient platelets were stimulated at 37°C for 5 minutes with either AYPGKF (500 μM) or thrombin (Thr; 0.5 unit/mL) without stirring. The addition of 2-MeSADP (1 μM) or epinephrine (10 μM) was made as indicated. The reaction was stopped by the addition of 3 × SDS sample buffer. Samples were separated by SDS-PAGE, Western blotted, and probed with anti-phospho-Akt (Ser⁴⁷³) antibody. The data shown are representative of 3 experiments. (C) Concentration-dependent Akt phosphorylation induced by combined G_12/13 and G_i signaling in Gα_q-deficient platelets. Gα_q-deficient platelets were stimulated at 37°C for 5 minutes with different concentrations of AYPGKF in the presence of 2-MeSADP (1 μM). The reaction was stopped by the addition of 3 × SDS sample buffer. Platelet proteins were separated by SDS-PAGE, Western blotted, and probed with anti-phospho-Akt (Ser⁴⁷³) antibody.