EpoR interacts with TfR2 in hematopoietic cells. (A) EpoR and TfR2 interact in UT7 cells. Left panel, UT7 cells were stimulated or not with biotinylated Epo (bEpo; 10 U/mL) for 10 minutes. After solubilization, the proteins were precipitated with streptavidin beads. The precipitated proteins and total cell lysates were analyzed by Western blotting using anti-TfR2 and anti-EpoR antibodies. Middle panel, UT7 cells were stimulated or not by Epo. After solubilization, proteins were immunoprecipitated using anti-EpoR or control antibodies and analyzed by Western blotting using anti-TfR2 antibodies. Right panel, UT7 cells were stimulated or not by Epo (10 U/mL). After solubilization, proteins were immunoprecipitated using anti-TfR2 or control antibodies and analyzed by Western blotting using anti-EpoR antibodies. (B) Association between EpoR and TfR2 in human erythroid progenitors. Primary human erythroid progenitors were produced from circulating hematopoietic progenitors using a 2-step culture protocol. Left panel, determination of TfR2 and EpoR expression in CD36⁻ cells and during erythroid differentiation of the CD36⁺ population. Right panel, on day 6 after CD36 cell sorting, CD36⁺ cells were harvested and solubilized. Proteins were immunoprecipitated using anti-EpoR antibodies or anti-TfR2 antibodies and then analyzed by Western blotting. (C) Association between EpoR and TfR2 in transfected HEK293 cells. HEK293 cells were transfected by expression vectors encoding TfR2, EpoR, or both. Expression of TfR2 and EpoR was analyzed by Western blotting using total cell lysates. After solubilization, proteins were immunoprecipitated using anti-EpoR or anti-TfR2 antibodies and analyzed by Western blot. (D) Cross-linking of ¹²⁵I-Epo in UT7 cells with or without sh TfR2 expression. UT7 cells expressing control shRNA or TfR2 shRNA were stimulated for 10 minutes with ¹²⁵I-Epo and treated with DSS to cross-link the proteins. After solubilization, EpoR was immunoprecipitated (panel 1). Immunoprecipitates from sh control cells were then submitted to denaturation by boiling in electrophoresis sample buffer, followed by a second immunoprecipitation using TfR2 antibodies (panel 2). Proteins were subsequently analyzed by polyacrylamide gel electrophoresis and autoradiography.³²  The exposure times were 24 hours and 10 days for panels 1 and 2, respectively.