Loss of Stat5 directly decreases IRP-2 gene expression. (A) Sequence of the IRP-2 promoter −1030 bp to −1100 bp upstream of the transcription start, showing one perfect GAS site (boxed in black) and 2 GAS sites with one mismatch (boxed in gray). (B) Multiple perfect Stat5 sites taken from Yao et al²⁶  and Ehret et al⁶⁵  together with the IRP-2-I. (C) Luciferase reporter assay using a DNA fragment ranging from 2 kb immediately upstream of the predicted IRP-2 transcription start site. Vertical lines indicate the approximate positions of the putative Stat5 response elements. 293T cells were cotransfected with constructs encoding IRP-2-firefly-luciferase, renilla-luciferase, Stat5a, and EpoR. Cells were treated with 10 U/mL Epo or left untreated, and luminescence was scored 3 hours later. Transfection efficiencies were normalized to renilla-luciferase activity (representative experiment; error bars are SD of experimental triplicates). (D) Epo-dependent induction of endogenous IRP-2 and oncostatin M analyzed via quantitative PCR in murine erythroid leukemia cells serum deprived for 3 hours, followed by stimulation with 10 U/mL Epo (1 hour). Quantitative PCR was normalized on HPRT (data are presented as means ± SD; n = 4). (E) 293T cells were cotransfected with constructs for EpoR and wt Stat5a, followed by 30 minutes of stimulation with 10 U Epo/mL. Whole-cell extracts of these cells were subjected to EMSAs using either the IRP-2-I oligonucleotide (left) or β-casein oligonucleotide as positive control (right). Respective arrows indicate Stat5 DNA complexes and Stat5 DNA complex supershifts. (F) Primary wt fetal liver erythroblasts were stimulated with Epo for 30 minutes, and ChIP for Stat5 was performed. Recovered DNA was analyzed for the presence of promoters of IRP-2 and CIS (positive control) by PCR. *P < .05; **P < .01.