HCP associates constitutively with the EPO receptor in SKT6 cells but is not tyrosine phosphorylated in response to EPO. (A) SKT6 cells were exposed to EPO at the concentrations and intervals described in the Materials and Methods and Results. JAK2, EPO receptors, and Stat5a then were immunoprecipitated from Triton-X 100 lysates, and levels of tyrosine phosphorylation of these factors were assayed by Western blotting with antibodies to phosphotyrosine (α-PY). (B) SKT6 cells were exposed to EPO (±50 U/mL) for 7.5 minutes and cell lysates were prepared as detailed in the Materials and Methods. HCP and EPO receptor complexes then were isolated by immunoprecipitation (IP) and were detected by Western blotting (W. blot). In the upper panel, the coimmunoprecipitation of HCP with EPO receptor complexes is shown. This association was constitutive, was observed in three independent experiments, and was not detectably modulated by EPO exposure. In the lower panel, levels of tyrosine phosphorylation of HCP in control versus EPO-stimulated cells were compared via the Western blotting of HCP immunoprecipitates with the α-PY antibody, 4G10.