Lyn and PLC-β3 regulate SHP-1 phosphorylation at Tyr⁵³⁶ and Tyr⁵⁶⁴. (A) Scheme of a panel of SHP-1 retroviral constructs. (B) CD34⁻KSL cells from me^v/me^v mice were retrovirally transduced with empty vector or SHP-1 mutants. GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF for 4 weeks and were analyzed by immunoblotting. Right 2 lanes indicate results with nontransduced cells. (C) BM cells were analyzed by flow cytometry for phospho-Tyr⁵³⁶ and phospho-Tyr⁵⁶⁴. MFI of SHP-1 phosphorylation in KSL cells was shown (n = 6). Data are mean ± SD, *P < .05 versus wt. (D) BMMCs were analyzed by immunoblotting. (E) wt CD34⁻KSL cells were retrovirally transduced with PLC-β3 CT. GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF for 4 weeks. Interactions among PLC-β3, Lyn, and SHP-1 were examined by coimmunoprecipitation followed by immunoblotting. (F) Left, PLC-β3^−/− CD34⁻KSL cells were retrovirally transduced with PLC-β3 CT. GFP⁺ cells were sorted and cultured in the presence of IL-3 and SCF for 4 weeks. The interaction among PLC-β3, Lyn, and SHP-1 was examined by coimmunoprecipitation followed by immunoblotting. Right, splenocytes were used for coimmunoprecipitation and immunoblotting. (G) GST-SHP-1 (0.5 μg) was incubated with various amounts of recombinant Lyn protein and analyzed by immunoblotting. (H) me^v/me^v BMMCs expressing the indicated SHP-1 proteins were immunoprecipitated with anti–SHP-1 and followed by immunoblotting.