Identification of C/EBPα-binding sites in the PU.1 distal enhancer. (A) Gel shift assay was carried out using 10 μg nuclear extract from 293T cells transfected with CMV-C/EBPα and 1 ng radiolabeled NE-C/EBP probe. Where indicated, 5 ng or 25 ng unlabeled NE or C1 to C5 double-stranded oligonucleotides were added as competitors (comp.) prior to addition of radiolabeled probe. Data with C5 are from a separate gel. (B) Gel shift assay was carried out using 10 μg nuclear extract from 293T cells transfected with CMV (−) or CMV-C/EBPα (α) and 1 ng of the indicated wild-type and mutant radiolabeled C/EBP site probes. See “Materials and methods” for a description of the point mutations in mC1, mC2, and mC5. (C) Gel shift assay was carried out using 12 μg nuclear extract from U937 cells and 1 ng radiolabeled C2 or C5 probes. Rabbit Ig (Ig), C/EBPα (α) or C/EBPβ (β) antiserum, or 25 ng unlabeled wild-type (WT) C2 or C5 or mutant (Mut) mC2 or mC5 competitor oligonucleotides were included in the gel shift reactions. The C/EBPα and C/EBPβ gel shift species and the supershifted species (*) are indicated.