Fig. 5. Effects of CE-WIB801C on inositol 1,4,5-trisphosphate receptor (IP3R) phosphorylation. Lane 1, Intact platelets (base); Lane 2, Collagen (10 μg/ml); Lane 3, Collagen (10 μg/ml)+CEWIB801C (200 μg/ml); Lane 4, Collagen (10 μg/ml)+CE-WIB801C (400 μg/ml); Lane 5, Collagen (10 μg/ml)+CE-WIB801C (400 μg/ml)+Rp-8-Br-cAMPS (250 μM); Lane 6, Collagen (10 μg/ml)+CE-WIB801C (400 μg/ml)+Rp-8-Br-cGMPS (250 μM); Lane 7, Collagen (10 μg/ml)+pCPT-cAMP (1 mM); Lane 8, Collagen (10 μg/ml)+8-BrcGMP (1 mM). Washed platelets (108/ml) were preincubatedwith or without W-cordycepin, the A-kinase inhibitor Rp-8-Br-cAMPS or the G-kinase inhibitor Rp-8-Br-cGMPS, and A-kinase activator pCPT-cAMP or the G-kinase activator 8-Br-cGMP in the presence of 2 mM CaCl2 for 3 min at 37°C, then stimulated with collagen (10 μg/ml) for 5 min at 37°C in an aggregometer. The reactions were terminated by adding an equal volume (250 μl) of lysis buffer. Proteins were separated by SDS-PAGE, transferred to PVDF, and immunoblotted with the indicated corresponding antibodies. The data are expressed as the mean ± S.E.M. (n=3). **p<0.001 versus the collagen-stimulated platelets, †p<0.05 versus the collagen-stimulated platelets in the presence of CE-WIB801C (400 μg/ml).
© Biomolecules & Therapeutics
