Effects of PKG and PKA inhibitors on 8-bromo-cGMP–induced phosphorylation of ERK MAP kinase. Washed platelets (1 × 10⁹/mL) were preincubated with PKG inhibitors Rp-pCPT-cGMPS (500 μM) or KT5823 (5 μM), or PKA inhibitors KT5720 (5 μM) or H89 (50 μM) for 10 minutes. Platelets were also preincubated with buffer or the same concentration of DMSO (0.25%) as controls. DMSO had no effect on ERK phosphorylation level in resting platelets in the absence of cGMP analogs (data not shown). Platelets were then stimulated for 30 seconds in the platelet aggregometer with (A) 8-bromo-cGMP (0.1 mM), (B) 8-pCPT-cGMP, or (C) NO donor, glyco-SNAP1 (1μM), solubilized in SDS-PAGE sample buffer, separated by SDS-PAGE, and immunoblotted with a rabbit antibody specific for phosphorylated Thr²⁰²/Tyr²⁰⁴ site of ERKs. Pictures shown in the figure are representative of 3 independent experiments. The bar graph depicts the quantitative results from 3 experiments obtained by scanning and quantifying the immunoblotting results using NIH Image software. To quantify the relative effects of PKG or PKA inhibitors on cGMP-induced ERK phosphorylation, the level of ERK phosphorylation induced by the cGMP analog was set as 100%.