Interaction analysis with the wild-type or mutated EpoR receptor baits using relay MAPPIT. (A) Expression of the wild-type EpoR or EpoR Y→F mutants in transiently transfected Hek293-T cells was verified by FACS analysis. The filled and open curves show parental and transfected Hek293-T cells, respectively. (B) Hek293-T cells were transiently cotransfected with vectors expressing a prey, the pXP2d2-rPAP1-luci reporter construct, and vectors expressing the wild-type EpoR or an EpoR mutant as indicated. After transfections, cells were stimulated with Epo or were left untreated (NS, not stimulated). Average values for relative luciferase activities (x-fold increase, luciferase values obtained from stimulated cells with respect to values derived from untreated cells) are shown. Representative experiments from at least 3 are shown. (C) Hek293-T cells were transfected with vectors expressing the EpoR, different preys, and the pXP2d2-rPAP1-luci reporter. After transfection, cells were stimulated with Epo or were left untreated (NS, not stimulated). Average values for relative luciferase activities (x-fold increase, luciferase values obtained from stimulated cells with respect to values derived from untreated cells) are shown. A representative experiment from 3 is shown.