sGC deficiency does not affect agonist-induced calcium mobilization, but inhibits Akt and ERK phosphorylation in response to either thrombin or collagen. (A-C) Washed platelets from wild-type (sGC^+/+; A) or sGC β₁ deficient mice (sGC^–/–; B) were labeled with 12.5μM Fura-2/AM/0.2% Pluronic F-127 and resuspended in Tyrode's solution. Platelets were then stimulated with selected concentrations of thrombin. Changes in intracellular free calcium concentrations were measured every 2 seconds and expressed as a ratio of fluorescence (FL) detected at 509 nm emission with an excitation wavelength of 340 nm and 380 nm. Statistical data from 3 experiments were shown in panel C. (D-E) Washed platelets from wild type (sGC^+/+) or sGC β₁ deficient mice (sGC^–/–) mice were stimulated with either collagen (D) or thrombin (E) at 37°C for 5 minutes and solubilized in 2× SDS sample buffer. Phosphorylation of Akt was detected by Western blotting with a rabbit monoclonal antibody specifically recognizing the phosphorylated Akt residue Ser⁴⁷³. A rabbit polyclonal antibody against nonphosphorylated Akt was used to verify equal loading. Data shown are representative of 3 independent experiments. (F) Washed platelets from wild type (sGC^+/+) or sGC β₁ deficient mice (sGC^–/–) mice were pre-incubated with 2MeSAMP (10μM) or buffer for 5 minutes, and then stimulated with thrombin at 37°C for 5 minutes and solubilized in 2× SDS sample buffer. Phosphorylation of Akt and ERK was detected by Western blotting using phospho-specific antibodies against phosphorylated Ser473 (Akt) and Thr-202/Tyr204 (ERK; Cell Signaling Technologies). A mouse monoclonal antibody against β-actin (Sigma-Aldrich) was used to verify equal loading.