Epo induces EpoR co-localization with epsin-1. (A) Endogenous epsin-1 becomes co-localized with wild-type EpoR upon Epo stimulation. Nonpermeabilized γ2A/HA-EpoR/JAK2 cells were stained with anti-HA antibodies to label the exofacial HA-tag of the EpoR as described previously.⁵  Cell were then treated with Epo for 12 minutes, fixed, permeabilized, immunostained for epsin-1, and visualized by confocal microscopy. The scale bar represents 5 μm. Representative co-localization areas are marked with arrows. (B) Epo induces p85 binding to epsin-1. ECFP-tagged epsin-1 and T7-tagged p85 were transiently expressed in γ2A/HA-EpoR/JAK2. Anti-T7 immunoprecipitates were immunoblotted with anti-GFP antibody to detect epsin-1. (C) EpoR-epsin-1 co-localization is impaired in cells expressing a ligase-deficient Cbl mutant. γ2A/HA-EpoR/JAK2 cells were transfected with either wild-type myc-tagged Cbl or Cbl(ΔY368). Forty-eight hours after transfection, cells were stained as described before, except Alexa 488 anti-myc antibody was used to mark transfected cells. The blue arrows indicate the transfected cells, and the white arrow shows co-localization in an untransfected cell. (D) Expression of epsin-1(ΔUIM) impaired EpoR internalization. Epo-induced EpoR internalization was determined by flow cytometry in γ2A cells co-transfected with vectors expressing HA-EpoR, JAK2, and ECFP-tagged epsin-1 (either wild-type or the ΔUIM mutant). Data were normalized to EpoR surface expression of unstimulated control cells. *P < .05 between epsin-1(WT) and epsin-1(ΔUIM). (E) BaF3 cells expressing HA-EpoR and epsin-1 (either wild-type or the ΔUIM mutant) were grown in RPMI media containing 2% fetal bovine serum (FBS) with different concentrations of Epo. Cell growth was measured using MTT assays. Cells grew similarly in WEHI media and 10% FBS. Epsin-1(ΔUIM)–expressing cells exhibit Epo hypersensitivity. **P < .005.