Cell-surface expression of TfR-1 is strongly reduced in Stat5^−/− erythroid progenitors. (A) Representative flow cytometry histograms of E13.5 wt and Stat5^−/− fetal liver cells stained for the erythroid markers TfR-1 and Ter-119 (left). The sequence from gate R1 (TfR-1^low Ter-119^low) to gate R5 (TfR-1⁻ Ter-119^high) represents development from the most immature erythroid progenitors (late BFU-E; CFU-E) to mature erythroid cells (orthochromatic erythroblasts; reticulocytes).⁵⁸  Quantification of gates R1 to R5 (data are presented as means ± SD; n = 4) (right). (B) Cell-surface expression of TfR-1 of Ter-119^high gated wt (dark gray line) or Stat5^−/− (light gray line) fetal liver cells (left). Quantification of TfR-1 cell-surface expression of wt and Stat5^−/− fetal livers (right; data are presented as means ± SD; n = 4). (C) Expression of TfR-1 mRNA from lysates of freshly isolated wt or Stat5^−/− fetal liver cells (data are presented as means ± SD; n = 3). Expression was normalized on hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels. (D) Western blot analysis of freshly isolated wt and Stat5^−/− fetal liver cell lysates for TfR-1. ERK was used as loading control. (E) Densitometric quantification of TfR-1 Western blot in 3D (data are presented as means ± SD; n = 4). (F) Primary wt fetal liver erythroblasts were stimulated with Epo for 30 minutes and ChIP for Stat5 was performed. DNA from Epo-stimulated primary wt erythroblasts was recovered using 2 different antisera directed against N- or C-terminal epitopes (αS5 C, αS5 N). Specific PCR products from Stat5-binding sites GAS 1, GAS 2, and GAS 3 in TfR-1 intron 1⁵⁹  were only obtained with Stat5-specific antibodies but not with control IgGs. PCR for the genuine Stat5 site in the CIS promoter was used as positive control. (G) Primary wt fetal liver–derived erythroblasts were factor depleted for 2.5 hours, followed by 1.5 hours of Epo stimulation (10 U/mL). TfR-1 mRNA expression was scored by qPCR normalized on HPRT (data are presented as means ± SD; n = 4). (H) Iron concentration in freshly isolated fetal liver lysates determined via atomic absorption spectrometry (data are presented as means ± SD; n = 4; for experimental details, see Document S1). *P < .05; **P < .01.