Lyn associates with the tyrosine-phosphorylated EpoR in hematopoietic cell lines. (A) 32D/EpoR-Wt cells, a clone of IL-3–dependent 32D cell line expressing the transfected wild-type murine EpoR cDNA, were starved overnight and left unstimulated (−) or stimulated with 100 U/mL of Epo (Ep) or 25 ng/mL of IL-3 (IL3) for 5 minutes at 37°C before solubilization. Cell lysates were immunoprecipitated with anti-Lyn. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblotting with an antiphosphotyrosine monoclonal antibody, 4G10 (αPY). (B) A human erythroleukemic cell line, F36E, was starved overnight and stimulated with 100 U/mL Epo for the indicated times. Cells were then lysed and analyzed as described above. (C) 32D/EpoR-Wt cells were left unstimulated or stimulated with Epo, as indicated. Cells were then lysed and immunoprecipitated with anti-Lyn. Anti-Lyn immunoprecipitates were then subjected to a second immunoprecipitation with anti-EpoR, anti-SHP-2, or preimmune serum (PI), as indicated, and analyzed by antiphosphotyrosine (αPY) immunoblotting. A 72-kD phosphotyrosyl protein that coimmunoprecipitates with Lyn from Epo-stimulated cell lysates is indicated with an asterisk. The positions of EpoR, Lyn, and the Ig heavy chain (IgH) are indicated. The molecular weight markers are indicated and given in kilodaltons.