Detection of bait-prey interactions with the different EpoR motifs. Isogenic populations of Hek293-Flp-In cells were selected expressing the different EpoR-derived tyrosine motifs as bait. FACS analysis (inset) shows the expression of the different chimeric bait receptors. Filled curves represent the parental cell line; open lines, the isogenic cell population stably expressing the chimeric bait receptor. Prey constructs are indicated at the left of each panel. These preys consist of the full-length or SH2 domains of signaling proteins that are fused to the C-terminal part of gp130. Hek293-Flp-In cells, stably expressing a single bait, were transiently cotransfected with the pXP2d2-rPAP1-luci and a prey construct as indicated. The full-length SV40T prey was used as a negative control; the PI3-K p85(2 × SH2) prey served as the positive control. This prey interacts with the LR-F3 domain as shown in the upper left panel. 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. The values obtained with the SV40T prey and the Y344 and Y402 bait were, respectively, 5- and 2-fold, due to a weak, direct interaction of STAT3 with these pY motifs. The values shown are normalized for this background. Representative experiments from at least 3 are shown in each case.