Phosphorylation and selective agonist-induced dephosphorylation of SPL Y398 and Y483. (A) Lysates from human platelets incubated with SFLLRN were precipitated with anti-SPL, then probed with a phosphotyrosine-specific Ab (4G10). The graph summarizes 3 studies (mean ± SEM). (B) Platelets were incubated with SFLLRN (N = 2-5) or U46619 (N = 4). Lysates were precipitated with 4G10 or a matched monoclonal control (Ig) and probed for SPL. (C) CHO cells were transfected with Myc-SPL (177-817) and SHP-1. Thrombin was added after an overnight incubation in serum-free medium beginning ∼ 30 hours after transfection (N = 4). (D) Platelets were incubated with collagen or ADP in the presence of ASA and/or apyrase as indicated (N = 2). (E) Phosphorylation sites. (Left) CHO cells were transfected with Myc-tagged, full-length WT SPL, or with Y398F, Y483F, and Y398/483F SPL variants. Lysates were precipitated with anti-Myc and probed for pTyr with 4G10 (N = 2). (Right) A representation of full-length SPL with all 14 tyrosine residues indicated. The sequence that includes Y398 and Y483 is shown. See supplemental Figure 1 for a map of protein binding domains that have been identified in SPL.