SCF causes tyrosine phosphorylation of EPO-R, but cannot replace EPO to support human erythropoiesis in vitro. Phosphorylation of c-Kit (A) and EPO-R (B) after stimulation of ECFC with EPO, SCF, or EPO + SCF. Replicate day-8 ECFC (∼5 × 106) were starved and then treated with SCF and EPO separately or in combination for 5 or 10 minutes as described in Materials and Methods. Cell extracts were subject to immunoprecipitation with anti–c-Kit and anti–EPO-R antibodies in the presence of protein A-Sepharose beads. Immunoprecipitates (corresponding to whole cell extracts containing 50 μg of total protein) were subjected to SDS-PAGE and Western blot analysis with antiphosphotyrosine antibody 4G-10. (C) To determine the requirement of SCF and EPO for growth of ECFC, day-6 ECFC (5 × 103) were incubated in 20% FBS, 1% BSA-containing medium supplemented with SCF, EPO, or SCF + EPO. Cell numbers were determined after 7 days in culture. Data represent the mean values from three independent experiments.
