Enforced activation of STAT5 in human CB CD34⁺ cells results in down-modulation of C/EBPα expression. (A) CB-derived CD34⁺ cells were transduced with MiGR1 or STAT5A^1*6, sorted on the basis of GFP expression, and total RNA was isolated and used in RT-PCR reactions with primers for C/EBPα and β-actin as indicated. (B) Schematic representation of the retroviral constructs that were used in these studies. (C) C/EBPα and C/EBPα-ER were overexpressed in 293T cells, and total cell lysates were Western blotted using anti-C/EBPα antibodies. (D) C/EBP-ER was overexpressed in 293T cells, and nuclear extracts were used in EMSA experiments as indicated, using a probe that contains the C/EBPα consensus binding sequence. 4-OHT induced DNA binding of C/EBPα-ER, which could be outcompeted with 50 × cold self-oligo (lane 4), while DNA binding was blocked by adding anti-C/EBPα (lane 6). (E) Luciferase assays were performed in 293T cells using luciferase reporters containing C/EBPα-binding sites. Cells were transfected with empty MiGR1 vector (control) or C/EBPα-ER expression vectors, and cells were either left unstimulated or were stimulated with 4-OHT. After 24 hours of stimulation, cells were harvested to perform luciferase and LacZ assays as described in “Materials and methods.”