Dose-dependent activation and time-dependent activation of Cbl tyrosine phosphorylation in DA-3-EPO-R cells. (A) DA-3-EPO-R cells were depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1, 7, and 13), or various concentrations of IL-3 (lanes 2-6 and 14) or EPO (lanes 8-12 and 15) for 15 minutes as shown. Following cell lysis, an immunoprecipitation was conducted with anti-Cbl polyclonal antibody. Lysate controls corresponding to 50 U/mL stimulation are shown in lanes 13-15. Western blot analysis using the monoclonal anti-phosphotyrosine 4G10 antibody was performed (pTyr immunoblot). Molecular mass standards are indicated. (B) DA-3-EPO-R cells were depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1 and 8), 50 U/mL IL-3 (lanes 2-7), or 50 U/mL EPO (lanes 9-14) for various times as shown. Following cell lysis, an immunoprecipitation was conducted with anti-Cbl polyclonal antibody. Western blotting using the monoclonal anti-phosphotyrosine 4G10 was performed (pTyr immunoblot). The membrane was then stripped and reprobed with a Cbl polyclonal antibody. Molecular mass standards are indicated.