(A) Coimmunoprecipitation of EpoR with β. Ba/F3, Ba/F3-EpoR, and Ba/F3-EpoR+ βc cells were incubated with Epo, and the solubilized proteins were immunoprecipitated with anti-EpoR (n-terminus), anti-β, or control antibody. After gel electrophoresis the immunoprecipitated proteins were transferred to nitrocellulose and blotted with antibody against EpoR c-terminus. Ba/F3-EpoR immunoprecipitated with antibody against β (lane 1), antibody against EpoR (lane 2), or control antibody (lane 3). Ba/F3 immunoprecipitated with anti-EpoR (lane 4). Ba/F3-EpoR+ βc immunoprecipitated with antibody against β(lane 5). Bands corresponding to EpoR were observed when EpoR expressing cells were immunoprecipitated with antibody against either EpoR or β. (B) Coimmunoprecipitation of β with EpoR. Ba/F3-EpoR+ βc cells were cultured without growth factor for 8 hours and then incubated in medium alone (lanes 1, 2, and 4) or medium containing Epo (50 U/mL) for 15 minutes (lane 3). The solubilized proteins were immunoprecipitated with either antibodies to the n-terminus of the EpoR (lanes 1 to 3) or monoclonal antibody to β (lane 4). After SDS-PAGE (7.5% acrylamide), the separated proteins were transferred to nylon membrane and immunoblotted with EpoR antibody (lane 1) or with polyclonal rabbit antibody against βc (lanes 2, 3, and 4). Bands corresponding to β were observed to similarly coimmunoprecipitate with EpoR in the presence or absence of Epo.
