Role of Ser 462 of the cytoplasmic domain in Epo/Epo-R routing to lysosomes. (A) BaF3 cells expressing Epo-R^WT or Epo-R^S462A were preincubated for 45 minutes with radiolabeled Epo. Cells were washed to remove free iodinated Epo; Epo bound at the cell surface and internalized were then determined. The cells were resuspended in culture medium containing an excess of unlabeled Epo to avoid rebinding of dissociated¹²⁵I-Epo molecules and incubated at 37°C. At the indicated times, cells were sampled and radioactivity present at the cell surface and inside the cells was measured as previously described.¹³  BaF3 cells expressing Epo-R^WT had accumulated 23,600 cpm/10⁶ cells inside the cells after 45 minutes of preincubation, while BaF3 cells expressing Epo-R^S462A had accumulated 29,300 cpm/10⁶ cells in the reported experiment. (B) Degradation of¹²⁵I-Epo released in the culture medium was tested by TCA precipitation with soluble radioactivity corresponding to degraded Epo. Experiments were performed in the absence (full lines) or in the presence (dotted lines) of methylamine (MeNH2).