Coimmunoprecipitation and phosphorylation of c-kit and EPO-R in HCD57 cells. (A) Stimulation with MR and soluble SCF. Factor-starved HCD57 cells were cocultured with mitomycin C–treated stromal cells expressing either the soluble (Sl/Sl⁴-S) or the MR (Sl/Sl⁴-MR) form of SCF for various periods of time at 37°C as described in Materials and Methods. Subsequently at various times, cell lysates were collected and subjected to IP with an anti–c-kit antibody and WB analysis with an anti-phosphotyrosine antibody and the enhanced chemiluminescence detection system. Coimmunoprecipitated and tyrosine-phosphorylated Golgi-processed c-kit (slow migrating)27 and EPO-R are indicated. Lane 1 corresponds to parental Sl/Sl4 cells cocultured for 10 minutes; lanes 2, 4, and 6 correspond to Sl/Sl⁴-S cells cocultured for 10, 60, and 120 minutes, respectively; lanes 3, 5, and 7 correspond to Sl/Sl⁴-MR cells cocultured for 10, 60, and 120 minutes, respectively. (B) Stimulation with EPO. Factor-starved HCD57 cells were exposed to no growth factor (lane 1) or stimulated with 10 μ/mL rhEPO for 5 minutes (lane 2) and for 10 minutes (lane 3). Cell lysates were subjected to IP using an anti–EPO-R antibody and WB using an anti-phosphotyrosine antibody as described above. Tyrosine-phosphorylated EPO-R is indicated.