Analysis of IRAG phosphorylation in intact human platelets with pSer664-IRAG and pSer677-IRAG antibodies. (A-B) Phosphorylation of IRAG stimulated in human platelets by 8-pCPT-cGMP (100 μM, 30 minutes) or by DEA/NO (10 μM, 1 minute). Reactions were preincubated with the cGMP kinase inhibitor Rp-8-Br-PET-cGMPS (200 μM, 20 minutes) where indicated. IRAG phosphorylation was analyzed by pSer664-IRAG antibody (A) or pSer677-IRAG antibody (B). Control conditions were performed for 8-pCPT-cGMP by adding H₂O and for DEA/NO by adding a final concentration of 1 mM NaOH to the reaction. (C, top) Kinetics of IRAG phosphorylation in human platelets by 8-pCPT-cGMP or by DEA/NO. IRAG phosphorylation was analyzed by pSer664-IRAG antibody. (C, bottom) Statistics of phosphorylation results in percentage of maximal Ser664 phosphorylation. Phosphorylation of VASP analyzed by the phosphospecific pSer239-VASP antibody and immunodecoration with IRAG- and VASP-specific antibodies are shown for comparison.