Figure 4
Role of Ser 462 of the cytoplasmic domain in Epo/Epo-R routing to lysosomes. (A) BaF3 cells expressing Epo-RWT or Epo-RS462A 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-RWT had accumulated 23,600 cpm/106 cells inside the cells after 45 minutes of preincubation, while BaF3 cells expressing Epo-RS462A 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).

Role of Ser 462 of the cytoplasmic domain in Epo/Epo-R routing to lysosomes. (A) BaF3 cells expressing Epo-RWT or Epo-RS462A 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-RWT had accumulated 23,600 cpm/106 cells inside the cells after 45 minutes of preincubation, while BaF3 cells expressing Epo-RS462A 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).

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