FIG. 6.

The effect of C/EBPβ and c-Jun interaction is DNA dependent. (A) Transcription factor c-Jun inhibits binding of C/EBPβ to the −100/−74 but not the −115/−74 oligonucleotide. TNFα promoter oligonucleotides −100/−74 (lanes 1 to 5) and −115/−74 (lanes 6 to 10) were ³²P labeled and used as probes in the gel shift assay. c-Jun (1 and 3 μg; lanes 2, 3, 7, and 8) and C/EBPβ (1.0 μl of baculovirus nuclear extract; lanes 1 and 7), alone or combined (lanes 4, 5, 9, and 10), were loaded on a 4% polyacrylamide gel and run under nonreducing conditions. (B) c-Jun and C/EBPβ form a complex binding to the −115/−74 TNFα promoter oligonucleotide. C/EBPβ (1 μl) and c-Jun (1 μg) were added alone (lanes 1 and 2, respectively) or together (lanes 3 to 6) to the ³²P-labeled −115/−74 oligonucleotide. Antibody to C/EBPβ (lane 4), control antibody (lane 5), or anti-c-Jun (lane 6) was added to the reaction mixture prior to loading on the gel. IgG, immunoglobulin G. (C) C/EBPβ alters the mobility of c-Jun bound to the −115/−98 TNFα promoter oligonucleotide. C/EBPβ (1 μl) and c-Jun (1 μg), alone (lanes 1 and 2) or together (lanes 3 to 5), were incubated with the ³²P-labeled −115/−98 oligonucleotide for 20 min at room temperature prior to running the gel. Antibodies to C/EBPβ and c-Jun (lanes 4 and 5) were incubated with the transcription factors for 20 min at room temperature prior to adding the radiolabeled oligonucleotide. (D) The proximity of AP-1 and C/EBPβ binding sites affects activation. A single copy of each of the AP-1 and C/EBPβ binding sites from the TNFα promoter was inserted into pT81-Luc either unchanged (0 bp) or with 10 irrelevant oligonucleotides (10 bp) separating the two sites. The plasmids (5 μg) were transfected by electroporation into wild-type U937 cells, keeping the total concentration of plasmid constant (16 μg). After transfection the cells were treated with PMA and LPS as described in the text. Cells were harvested and analyzed as described in Materials and Methods. The parent plasmid was transfected in each experiment, and the results were subtracted prior to analysis. The results presented as the means ± 1 SE are representative of four independent experiments.