Dissociation of the SPL/SHP1 complex in response to thrombin and TxA2 but not collagen or ADP. (A) Human platelets were incubated with SFLLRN or U46619 ± the SHP-1/SHP-2 inhibitor, NSC87877, and then precipitated with anti–SHP-1 before being probed for SPL (N = 4 for SFLLRN; 2 for U46619). (B) Human platelets were incubated with collagen or ADP ± ASA and apyrase as indicated in the figure, and then precipitated with anti–SHP-1 before being probed for SPL (N = 3 for collagen; 2 for ADP). (C) Lysates were prepared from CHO cells cotransfected with SHP-1 and Myc-SPL (N = 2). Proteins were precipitated with (top) anti-Myc or nonimmune globulin (Ig), and then probed with SHP-1 and Myc-SPL, or (bottom) precipitated with anti–SHP-1 and probed for Myc-SPL and SHP-1. (D) CHO cells were transfected with Myc-SPL and SHP-1. Thrombin was added after an overnight incubation in serum-free medium beginning ∼ 30 hours after transfection. Proteins were precipitated with (top) SHP-1, and then probed with Myc-SPL and SHP-1, or (bottom) were precipitated with anti-Myc and probed for SHP-1 and Myc-SPL (N = 3). (E) CHO cells were transfected with RGS18 and SHP-1 ± full-length SPL and then serum-starved. Proteins were precipitated with anti-RGS18 and probed for SHP-1 (N = 3). (F) CHO cells were transfected with SHP-1 and a Myc-tagged SPL fragment encompassing 376-586 in which Y398 and/or Y483 were replaced with Phe as indicated. Proteins were precipitated with anti-Myc and probed for SHP-1, after which time they were reprobed for Myc as indicated. (Left) Representative experiment. (Right) Summary of 3 experiments (mean ± SEM).
