C/EBPα regulates nfkb1 gene expression via 2 conserved κB sites. (A) Diagram of the murine nfkb1 promoter, which lacks a TATAA box. Locations of the indicated transcription factor binding sites are marked relative to the transcription initiation site. (B) NIH-3T3 cells were transiently cotransfected with 1 μg of nfkb1–1750-Luc or its truncated variants nfkb1–912, –528-Luc, or –257-Luc, 5 ng of CMV-β-Gal, and 100 ng of CMV, CMV-C/EBPα variants, or CMV-NF-κB p50. Fold activation of the full-length or truncated promoters relative to empty CMV plasmid was determined after adjustment for β-galactosidase activity. The averages from 3 independent experiments are shown. Expression level of the various C/EBPα variants was determined by Western blotting. (C) Fold activation of the nfkb1-Luc or (C/EBP)₂TK-Luc reporter by C/EBPβ or its LIP isoform, relative to empty CMV was determined by cotransfection in HeLa cells as described earlier. Luciferase and β-galactosidase activity was measured 48 hours after transfection, and the average corrected activity from 3 experiments is presented. (D) 293T cells were transiently transfected with 3 μg of CMV-C/EBPα, CMV-p50, or empty CMV vector as indicated. Nuclear extracts were subjected to gel shift analysis with the use of oligonucleotides corresponding to the κB sites from the nfkb1 promoter (κB1 or κB2), their corresponding mutants (mκB1 or mκB2), or a consensus C/EBP-binding site from the neutrophil elastase (NE) promoter. (E) NIH-3T3 cells were cotransfected with 1 μg of the nfkb1–257-Luc reporter or its variants carrying clustered point mutations of either one or both κB sites in the nfkb1 promoter (mκB1-, mκB2-, or mκB1+2-Luc, respectively) and with 100 ng of CMV, CMV-C/EBPα, or CMV-C/EBPαLZ and 5 ng of CMV-β-Gal. Corrected activation by the indicated C/EBPα isoform relative to empty CMV was determined, and the average activation ratio of each mutant relative to the WT reporter from 3 experiments is presented.