Dose-dependent activation and time course of Shc tyrosine phosphorylation in CTLL-EPO-R cells. (A) CTLL-EPO-R cells were incubated in the depleted of cytokine for 8 hours and then stimulated with no added factor (lanes 1 and 7), or various concentrations of IL-2 (lanes 2 through 6) or EPO (lanes 8 through 13) for 5 minutes as shown. After cell lysis, an immunoprecipitation was conducted with anti-Shc polyclonal antibody. Western blot analysis using the monoclonal antiphosphotyrosine 4G10 antibody was performed (pTyr immunoblot). The blot was stripped and reprobed with an anti-Shc polyclonal antibody (Shc immunoblot) or an anti-Grb2 MoAb (Grb2 immunoblot). Molecular mass standards are indicated. Ab, antibody. (B) CTLL-EPO-R subclone 22 (lanes 1 through 12) and CTLL-EPO-R subclone 5 (lanes 13 and 14) were starved for 8 hours and then stimulated with no added factor (lanes 1, 7, and 13), 50 U/mL IL-2 (lanes 2 through 6), or 50 U/mL EPO (lanes 8 through 12 and 14) for various times as shown. After cell lysis, an immunoprecipitation was conducted with anti-Shc polyclonal antibody. Western blotting using the monoclonal antiphosphotyrosine 4G10 was performed (pTyr immunoblot). The blot was stripped and reprobed with an anti-Shc polyclonal antibody (Shc immunoblot) or an anti-Grb2 MoAb (Grb2 immunoblot). Molecular mass standards are indicated. Ab, antibody.