SHP-1 activation links thrombin to dephosphorylation and dissociation of the pSPL/RGS/SHP1 complex. (A-B) Human platelets were incubated with SFLLRN ± NSC87877 to inhibit SHP-1 and then precipitated with the phosphotyrosine-specific Ab, 4G10 (A), or anti-RGS18 (B) before being probed for SPL. (C) CHO cells were transfected with Myc-SPL and SHP-1 (not tagged). Thrombin was added after an overnight incubation in serum-free medium beginning ∼ 30 hours after transfection. Phosphatase activity was measured in anti-Myc precipitates (mean ± SEM; N = 3). (D top) Similar to panel C except that the anti-Myc precipitate was probed with a pY536-specific SHP-1 Ab. (D bottom) Platelets were preincubated for 10 minutes with either the inhibitor of Src family kinases, PP2, or the inactive congener, PP3, and then stimulated with thrombin as indicated (N = 2). (E) Similar to panel C except that the cells were cotransfected with Myc-SPL and either SHP-1 or SHP-1 Y536F. SPL-associated phosphatase activity was measured (mean ± SEM; N = 3). (F) CHO cells were cotransfected with Myc-SPL and either WT SHP-1 or SHP-1 Y536F. The anti-Myc precipitate was probed for SHP-1 (representative of 2 experiments).