Identification of NF-Y as the transcription factor that binds to the −65/−52 IL-6RE of the JunB promoter.

M1 cells stimulated with IL-6 (100 ng/mL) for 15 minutes were used to prepare nuclear extracts. Protein-DNA complexes were analyzed by EMSA, as described in “Materials and methods.” (A) Oligonucleotide competition. The 100-fold excess unlabeled oligonucleotides were added to nuclear extracts 5 minutes before the addition of labeled JunBWT probe. Control sample had no oligonucleotide competitor. Arrow indicates the NF-Y-DNA complex. (B) Antibody competition. Antibodies (2-5 μg), unless otherwise indicated, were incubated with nuclear extract at RT for 30 minutes before addition of labeled JunBWT probe. Two different antibodies for C/EBP-b were used (lanes 4-7). NF-YA, NF-YB, and NF-YC antibodies (1 μg) were incubated with extract on ice for 2 hours before the addition of labeled probe. All antibodies were obtained from Santa Cruz.