Jak kinase(s) and c-Kit cooperate in cS5 activation. (A) Lysates from self-renewing WT GFP, WT cS5, Jak2^−/−-cS5, and EpoR^−/−-cS5 cultures were analyzed for P-Y-Stat5, total Stat5, Jak2, and EpoR protein levels. Actin, loading control. (B) cS5-expressing WT erythroblasts were starved for 3 hours (−) and subsequently stimulated with Epo, SCF, or Epo + SCF for 10 minutes. Lysates were analyzed for P-Y-Stat5 and total Stat5 protein. Erk1/2, loading control. (C) WT erythroblasts expressing GFP or cS5, and Jak2^−/− and EpoR^−/− cells expressing cS5 were starved for 3 hours (−) and subsequently stimulated with Epo, SCF, or SCF + imatinib (10 μmol/L) for 10 minutes. Lysates were analyzed for P-Y-Stat5 and total Stat5 protein. Erk1/2, loading control. (D) WT GFP, WT cS5, Jak2^−/−-cS5, and EpoR^−/−-cS5 cultures were starved for 3 hours (−) and subsequently restimulated with SCF for 2 hours (+). Expression of the Stat5 target genes oncostatinM, cyclin D2, and c-myc was assessed by real-time PCR.