Jak2 and Stat5 activation via EpoR-HM and EpoR-H alleles in primary bone marrow–derived erythroblasts. (A) Minimal EpoR alleles. Diagrammed are knocked-in PY-null EpoR-HM and PY343-encoding EpoR-H alleles, together with the wt-EpoR. (B) Jak2 activation profiles via minimal EpoR alleles. (Bi) Erythroid progenitor cells from wt-EpoR, EpoR-HM, and EpoR-H mice were expanded to yield (on day 3) 45% to 50% frequencies of CD71^high erythroblasts. Washed cells were cultured for 6 hours in the absence of hematopoietic cytokines (10 μg/mL transferrin, 10 ng/mL insulin, 0.5% BSA in IMDM). Cells were then exposed to Epo (2.5 U/mL), and at the indicated intervals lysates were prepared for Western blot analyses. For phospho-Jak2, note the fairly uniform activation profiles supported via EpoR-H, EpoR-HM, and wt-EpoR erythroblasts. In all expansion experiments, CD71 and Ter119 marker expression was assessed, and representative distributions are shown. (Bii) Jak2 activation was analyzed as above, except for erythroblast preparations from which Ter119^pos cells were depleted. The numbers in quadrants (clockwise from bottom left) indicate the percentage of CD71^negTer119^neg, Ter119^negCD71^pos, CD71^posTer119^high, and Ter119^highCD71^neg cells among total live-gated cells. (C) Stat5 activation via EpoR-H, but not EpoR-HM, alleles. In parallel analyses, EpoR allele–mediated activation of Stat5 was assessed both for expansion cultures (Ci) and for Ter119-depleted cultures (CD71^posTer119^neg populations) (Cii).