Effects of PKG and PKA inhibitors on 8-bromo-cGMP–induced phosphorylation of VASP at Ser²³⁹ in human platelets. Washed platelets (3 × 10⁸/mL) were preincubated with (A) PKG inhibitor KT5823 (5 μM), PKA inhibitor KT5720 (5 μM), or PKA inhibitor H89 (50 μM); and (B) PKG inhibitor Rp-pCPT-cGMPS (0.2 and 0.5 mM), or PKA inhibitor Rp-Br-cAMPS (0.2 and 0.5 mM); and (C) myristoylated PKI peptide (14-22) (5 μM) for 10 minutes. Platelets were also preincubated with buffer or DMSO as controls. Platelets were further incubated with 8-bromo-cGMP (50 μM) at 37°C in the platelet aggregometer for 10 minutes (shorter incubation with 8-bromo-cGMP failed to induce significant VASP phosphorylation) and solubilized directly in SDS-PAGE sample buffer. Phosphorylation of VASP at Ser²³⁹ was detected by immunoblotting with a monoclonal antibody specifically recognizing Ser²³⁹-phosphorylated VASP, 16C2. Pictures shown in the figure are representative of 3 independent experiments. The bar graph depicts the quantitative results from 3 experiments obtained by scanning the 16C2 reactive bands and quantifying optical density using NIH Image software.