Truncated EpoR mutants fail to bind p85β or internalize on Epo stimulation. (A) Schematic diagram for truncated EpoR mutants used in our studies based on those from PFCP patients. (B) Truncated EpoR mutants did not internalize on Epo treatment by flow cytometry in γ2A cells. (C) Epo-induced internalization was defective for Stop2, but not Stop2-KYLYL, in erythroid progenitor cells. (D) Stop2+KYLYL, but not Stop1, Stop2, or Stop2+KFLFL, interacted with p85β on Epo induction. (E) Truncated EpoR mutants are hypersensitive to Epo in Ba/F3 cells. Ba/F3 cells stably expressing wild-type or truncated EpoR mutants were grown under different Epo concentrations. Cell numbers were measured by MTT assays every 24 hours for 3 days. Cell numbers from cultures in WEHI media as a source of IL-3 were shown as controls. (F) Erythroid progenitor cells expressing Stop2, but not Stop2+KYLYL, are hypersensitive to Epo. Erythroid progenitor cells were transduced with retroviruses expressing wild-type or truncated EpoR mutants. At 24 hours after infection, cells were washed and cultured in media containing different Epo concentration and 2% FBS (top panel), and cell numbers were measured by MTT assays after 24 hours. Cell numbers from cultures in media containing 10% FBS and 2 U/mL Epo (Epo media) were shown as controls (bottom panel). IP indicates immunoprecipitation; IB, immunoblot; P-JAK2, phosphorylated active JAK2.