EpoR expression and rHuEpo-dependent JAK-2 activation in EA.hy926 cells. (A) Twenty-five–microgram aliquots of the extracts of confluent EA.hy926 cells and HUVECs were run on 8% SDS-PAGE gel and probed with anti-EpoR rabbit antiserum. EpoR synthetic peptide was used as a positive control. (B through D) EA.hy926 cells were incubated with 30 U/mL rHuEpo in serum-free conditions for the indicated periods of time. Cell extracts were then immunoprecipitated with anti–JAK-2 antibody. Immunoprecipitates were subjected to 8% SDS-PAGE and probed with anti-phosphotyrosine antibody in a Western blot (B). After stripping of the membrane, immunoprecipitates were probed with anti-EpoR antibody (C). Note that EpoR coprecipitates with phosphorylated JAK-2. Uniform loading of the gel was shown by probing the membrane with anti–JAK-2 antibody (D).