Altered Bcl-xl and DAPK-2 expression in EpoR-HM erythroblasts. (Ai) Levels of Bcl-xl in expanded wt-EpoR, EpoR-HM, and EpoR-H erythroblasts were assayed (by Western blotting) at 2 time points—directly following cytokine withdrawal and at 30 minutes of Epo exposure. Note the decreased Bcl-xl levels in EpoR-HM erythroblasts. For comparison, Bax levels also were analyzed. (Aii) Bcl-xl expression in wt-EpoR, EpoR-HM, and EpoR-H erythroblasts also was analyzed in Ter119-depleted, CD71^high erythroblasts. (B) Defective survival of EpoR-HM CD71^highKit^neg erythroblasts and rescue of survival potential by PY343 in EpoR-H. Kit^pos progenitor cells were prepared from wt-EpoR, EpoR-HM, and EpoR-H bone marrow and were expanded in SP34-EX media. At day 3 of culture, CD71 and Ter119 marker expression was assayed and cells were costained with annexin-V. Relative frequencies of annexin-V–positive cells among CD71^pos subpopulations of EpoR-HM, EpoR-H, and wt-EpoR erythroblasts are graphed. Expanded cells also were shifted to differentiation medium, and frequencies of annexin-V and Ter119–copositive cells were analyzed. (C) Elevated DAPK-2 expression in EpoR-HM erythroblasts. Death-associated protein kinase-2 (DAPK-2) expression in wt-EpoR, EpoR-HM, and EpoR-H erythroblasts was assayed by Western blotting (and digital densitometry). Note the several fold increase in DAPK-2 levels in EpoR-HM erythroblasts (representative of 3 independent experiments). For Bcl-xl, plotted quantitation values are mean values plus or minus SE; n = 3 wt-EpoR, EpoR-HM, and EpoR-H mice per group, x = 2 experiments. For annexin-V staining, plotted values are mean values plus or minus SE; n = 3 wt-EpoR, EpoR-HM, and EpoR-H mice per group, x = 4 experiments.