Lyn is involved in CD150 tyrosine phosphorylation. CD150-SHP-2 association in DT40 transfectants depends on Lyn. Anti-SHP-2 (A, top panel), anti-SH2D1A (lower panel), and antiphosphotyrosine (B, top panel) Western blots on CD150 immunoprecipitated with anti-CD150 mAb. The binding of SH2D1A to CD150 reduced the association of CD150 with SHP-2. The CD150-SHP-2 association was BTK-independent, completely depended on Lyn expression and CD150 tyrosine phosphorylation. Amount of CD150 in immunoprecipitates was controlled by surface biotinylation of CD150 (B, bottom panel). One of 5 experiments. (C) Lyn directly phosphorylates CD150. Lyn and Syk tyrosine kinases were immunoprecipitated from cell line MP-1, and cold in vitro kinase assays followed by antiphosphotyrosine Western blot were performed. GST fusion protein of CD150ct (GST-CD150ct) and MBP served as a substrate in in vitro kinase assays. One of 3 experiments. (D) Lyn predominately phosphorylates Y327 in CD150ct (MU). Cold in vitro kinase assays on Lyn immunoprecipitates were performed using the following substrates: GST and GST-CD150ct with point mutations Y281F (M2), Y307F (M3), Y327 (M4), and Y281/327F (M2M4). Antiphosphotyrosine Western blot (top panel) and anti-GST Western blot (bottom panel). Concentrations of GST-proteins based on anti-GST blot were taken into account for densitometry analysis of anti-phosphotyrosine Western blot (E). One of 3 experiments.