The expression of TfR2 increases the number of EpoR molecules at the cell surface. (A) Analysis of ¹²⁵I-Epo binding to HEK293 cells transfected with expression vectors for TfR2 (filled triangles), EpoR (empty squares) or both (filled squares) by the Scatchard method as previously described.²  The efficient expression of TfR2 and EpoR was verified by Western blotting (insert). (B) ¹²⁵I-Epo binding to HEK293 cells transfected or not (NT) with plasmids encoding TfR2, Jak2, and/or EpoR (ER). Cells were incubated for 10 minutes with ¹²⁵I-Epo and specifically bound radioactivity was measured. The histogram shows the means ± SD from 5 independent experiments. Insert presents the results of Student t test analysis: NS: not significant, *P < .05, **P < .01, ***P < .001. (C) TfR2 knockdown via shRNA reduces Epo binding to EpoR in UT7 cells. Aliquots of 2 × 10⁶ UT7 cells expressing control shRNA, TfR2 shRNA A, or TfR2 shRNA B were incubated for 10 minutes with ¹²⁵I-Epo and specifically bound radioactivity was then measured. The presented results correspond to 6 independent experiments; 100% correspond to ¹²⁵I-Epo specific binding in cells expressing a control shRNA and ranged from 11 450 cpm to 19 372 cpm in the 6 experiments. The inhibition of TfR2 expression was verified by Western blotting. Insert, (1) = sh Control; (2) = sh TfR2 A; (3) = sh TfR2 B. ** indicate a significant difference (P < .01) relative to cells transfected with the control shRNA. Difference between cells expressing shTfR2 A and shTfR2 B was not significant (P > .35). (D) Stability of the cell surface EpoR in UT7 cells expressing either a control or TfR2 shRNA construct. Epo-starved UT7 cells were incubated with 500μM cycloheximide for the indicated times and cell surface EpoRs were quantified after a 10-minute incubation with 1nM ¹²⁵I-Epo. (E-F) TfR2 and EpoR proteins interact at an early stage of the maturation process. UT7 cells were Epo-starved and incubated in the absence or presence of 5 μg/mL BFA for 15 hours. Cell viability was tested by measuring cellular ATP levels after that deprivation period and UT7 cells deprived for 3 days of Epo were used as control. EpoR cell surface expression was also tested using radiolabeled Epo in viable Epo-deprived cells (ND: not done; 3E). UT7 cells were solubilized, proteins were immunoprecipitated using anti-EpoR or anti-TfR2 antibodies, and analyzed by Western blotting (3F).