Loss of the antiapoptotic protein Mcl-1 in Stat5^−/− fetal liver. (A) Western blot of lysates from freshly isolated fetal livers or Ter-119⁺ and Ter-119⁻ subfractions (separated using magnetic beads; “Methods”) for Bcl-x_L (left; ERK, loading control). Western blot for Mcl-1 of 2 individual freshly isolated fetal livers of each genotype (right; eIF4E, loading control). (B) Quantitative PCR analysis for Mcl-1 mRNA isolated from the Ter-119⁺ subfraction of freshly isolated fetal liver cells (data are presented as means ± SD; n = 3). (C) wt and Stat5^−/− primary erythroblasts expanded for 5 days under self renewal conditions (“Methods”) were deprived of factors for 3 hours, followed by a 30-minute restimulation with 10 U/mL Epo. qPCR analysis for Mcl-1 (representative experiment; error bars are SD of experimental triplicates). (D) wt or Stat5^−/− fetal liver cells were infected with retroviruses encoding GFP alone (murine stem cell virus [MSCV]), or Bcl-x_L plus GFP, or Mcl-1 plus GFP from bicistronic constructs. After retroviral infection (72 hours), primary erythroblasts were cultivated for another 48 hours under self-renewal conditions (“Methods”) in the presence or absence of Epo. Rates of apoptosis were determined by flow cytometry for annexin V. One representative set of histograms from 3 independently performed experiments of GFP-gated annexin V⁺ cells at 48 hours of treatment is depicted. Arrowheads indicate increased levels of apoptosis. *P < .05; **P < .01.