p85α and p85β are important in mediating EpoR internalization. (A) Wortmannin treatment does not affect EpoR internalization. γ2A cells stably expressing HA-EpoR and JAK2 were treated with wortmannin for 2 hours at indicated concentrations followed by Epo stimulation. At 45 minutes after Epo stimulation, surface EpoRs were quantified by flow cytometry. Wortmannin inhibited AKT activation as detected by phospho-AKT antibodies. (B) Dominant-negative forms of p85α and p85β impair EpoR internalization. Epo-induced EpoR internalization was measured by flow cytometry in γ2A cells stably expressing HA-EpoR and JAK2 transiently transfected with vectors expressing the N or C terminal-SH2 domains from p85α and p85β. (1) vector control; (2) p85α N-terminal SH2; (3) p85α C-terminal SH2; (4) p85β N-terminal SH2; (5) p85β C-terminal SH2; (6) p85α and p85β N-terminal SH2s; (7) p85α and p85β C-terminal SH2s. *P = .001 (unpaired t test) versus control. (C) p85β binds EpoR on ligand stimulation. γ2A cells stably expressing HA-EpoR and JAK2 were transiently transfected with vectors expressing full-length p85β. At 48 hours after transfection, cells were starved overnight and treated with Epo for 10 minutes. Cell lysates were subjected to immunoprecipitation by p85β antibodies and immunoblotted with anti-HA antibody for the receptor. Cell lysates were also subjected to immunoblotting with antibodies to active JAK2 (P-JAK2), p85β, and HA. IP indicates immunoprecipitation; and IB, immunoblot.