C/EBPα blocks the transactivation capacity of PU.1.
(A) Transient transfection in 293T cells with a reporter construct of a minimal TK promoter with PU.1 binding sites only (p(PU.1)4TK) or mutated PU.1 DNA-binding sites only (p(mutated PU.1)4TK) and expression plasmids for PU.1, C/EBPα, and Gal4-VP16 or empty vector, respectively. As controls, a minimal TK promoter with C/EBP-binding sites (p(C/EBP)2TK) and a pGal4-luc reporter were used. Promoter activity was measured as luciferase activity 24 hours after transfection. (B) C/EBPα binds to PU.1 in vitro. For the protein interaction assay, (35S) Met-labeled in vitro–translated C/EBPα (lane 2) was incubated with 1 μg bacterially expressed GST-PU.1 (lanes 5, 6). Equivalent amounts of GST protein or glutathione agarose beads (lanes 3, 4) were incubated with in vitro–translated C/EBPα, and as a control, in vitro–translated c-Jun was incubated with GST-PU.1 in lane 1.
