Figure 2.

Activation of EPO-R signaling pathway by peptide derived from the homology domain. (A) Phosphorylation of JAK2. TF-1 cells were stimulated in RPMI serum-free medium for 30 min at 37°C with 1 unit/ml EPO or 10 μM ERP. After cell lysis, immunoprecipitation (IP) was done with anti-JAK2 antibody (αJAK2), followed by Western blot analysis (WB) with antiphosphotyrosine antibody, 4G10, (αPY, Upper) or anti-JAK2 antibody (αJAK2, Lower). Cells were stimulated with hormone or peptide as indicated at top. Panels are digital images of Western blots. Arrow at right indicates position of phosphorylated JAK2 (pJAK2). Lowest band on upper panels represents a protein phosphorylated in the absence of hormone or peptide. (B) Phosphorylation of EPO-R and STAT5. Cells were stimulated as for A. After lysis and IP with αPY99 antibody, proteins were separated on 8% SDS/PAGE, and WB was performed by using anti-EPO-R antibody (αEPO-R, Upper) or anti-STAT5 antibody (αSTAT5, Lower). Arrows at right indicate position of phosphorylated EPO-R (pEPO-R) and STAT5 (pSTAT5). Other symbols as in A. Cells were unstimulated or incubated with hormone or peptide as indicated. (C) Dose-response curve for activation of EPO-R signaling pathway by ERP. TF-1 cells were stimulated for 30 min at 37°C with ERP at concentrations shown. Cell lysis, IP, and quantification of JAK2 phosphorylation as described in A. Points are mean ± SEM of densitometry data of three independent experiments, expressed as percent of maximum stimulation in each experiment. EC₅₀ of 100nM ERP shown here may be compared with 1.5 units/ml EPO (not shown).