Cbl and its E3 ligase activity are essential for Epo-induced EpoR internalization. (A) siRNAs for Cbl or control siRNAs were transfected into γ2A/HA-EpoR/JAK2 cells, and levels of surface EpoR before and after 45 minutes of Epo stimulation were analyzed by flow cytometry. For each sample, EpoR surface expression was normalized to that from samples expressing control siRNA before Epo stimulation. Knockdown efficiency of Cbl is shown. Immunoblotting of actin was used as a loading control. (B) Cbl knockdown in BaF3 cells impairs Epo-induced EpoR internalization. Cells expressing shRNA for Cbl or control shRNA, identified by GFP expression, were gated for analyses. The numbers in parentheses are median fluorescence intensity, which represents EpoR surface expression. Representative histograms from 3 independent experiments are shown. (C) Epo-induced EpoR internalization is impaired in γ2A/HA-EpoR/JAK2 cells expressing ligase-deficient Cbl mutants (ΔY368 and ΔY371). (D) Epo-induced EpoR internalization is significantly impaired by the expression of dominant-negative Cbl in Ter119^– erythroid progenitor cells. *P < .05, **P < .005 (Student t test).