JAK2 tyrosine kinase activity is required for ligand-induced EpoR down-regulation. (A) Mature HA-EpoR species were detected in JAK2-deficient γ2A cells stably expressing HA-EpoR with wild-type or kinase-deficient JAK2 (JAK2KD) by their resistance to Endo H treatment (left). At 45 minutes after Epo induction, mature HA-EpoR was degraded, and this degradation requires JAK2 kinase activity (right). (B) HA-EpoR at the γ2A cell surface was detected by surface IP. On Epo induction, surface EpoR disappeared when coexpressed with JAK2 but not JAK2KD. (C) Cell-surface HA-EpoR was quantified by flow cytometry using APC-conjugated anti-HA antibodies in nonpermeabilized γ2A cells. Representative histograms of total receptor expression levels (GFP) and cell-surface receptor expression levels (APC) are shown. In each histogram, the uninduced sample is in gray and the induced sample is in black. Median fluorescence of each sample is in parentheses. (D) Kinetics of ligand-induced EpoR internalization. Levels of cell-surface HA-EpoR were analyzed by flow cytometry at various time points after induction as described in panel C. Immunoblotting with anti-JAK2 antibodies showed that the expression levels of JAK2 and JAK2KD are similar. All data represent results from at least 3 independent experiments. Endo H indicates endoglycosidase H; N + P, neuraminidase + PNGaseF; IB, immunoblot; and V, vector.