Forskolin and PGI2 cause dissociation of the SPL/RGS/SHP-1 complex, but do not affect SPL tyrosine phosphorylation. (A) Human platelets were incubated with 20 µM forskolin (Forsk) ± 1 µM okadaic acid as indicated. Proteins were precipitated with anti-RGS18 or nonimmune immunoglobulin (Ig) and then probed with anti-SPL before reprobing with anti-RGS18. The graph shows mean ± SEM. (B) Identical to panel A except 15 µM PGI2 was used instead of forskolin. Panels C and D are identical to panels A and B except that immunoprecipitation was performed with anti-SHP-1 instead of anti-RGS18. Panels A-C, N = 4; panel D, N = 3. (E) Human platelets were incubated with 20 µM forskolin ± 1 µM okadaic acid as indicated. Proteins were precipitated with the anti-phosphotyrosine antibody, 4G10, or nonimmune Ig and then probed with anti-SPL. The graph summarizes 4 experiments (mean ± SEM). Panel F is identical to panel E except 15 µM PGI2 was used instead of forskolin; N = 3. P values are relative to resting platelets.
