cGKI macrocomplex and IRAG phosphorylation in human platelets. (A) Identification of IRAG and cGKIβ in human platelets (P-M indicates platelet membranes; P-C, platelet cytosol) by immunoblot analysis with specific antibodies directed against IRAG, cGKIα, or cGKIβ. (B) Isolation and phosphorylation of the ternary complex of IRAG, cGKI, and InsP₃RI in human platelets. The complex was isolated using cGMP agarose beads and then phosphorylated by the addition of 8-pCPT-cGMP (3 μM) and [γ-³² P]ATP. ³² P-phosphorylation was analyzed by autoradiography (AR), and immunoblot analysis (IB) was performed with specific antibodies. The positions of molecular weight markers are indicated. Note that the cGKI substrate VASP was not assembled in the cGKI complex. (C) ³³ P-phosphorylation of IRAG in intact human platelets stimulated with the cGMP analog 8-pCPT-cGMP (100 μM) for 1 to 30 minutes. (D) Stimulation of human platelets with the nitric oxide donor GEA-NO (100 μM). As control, in each experiment the phosphorylation of IRAG was compared with that of Ser239-VASP, determined by immunoblot analysis with pSer239-VASP–specific antibodies. Equal amounts of total IRAG or VASP in the different lanes were checked by immunoblotting with IRAG- and VASP-specific antibodies (C-D). (E) Statistics of phosphorylation with 8-pCPT-cGMP.