CHOP inhibits the DNA binding of C/EBPϵ to the putative C/EBP site on the lactoferrin promoter, as shown by EMSA. (A) EMSA was performed using 10 μg nuclear extract proteins from untreated NB4 cells (NB4) or NB4 cells treated with ATRA for 24 hours (NB4A). Extracts were incubated with 32P-labeled oligonucleotides containing the C/EBP binding site from the lactoferrin promoter. Unlabeled competitor (cold oligo), C/EBPϵ antibody (anti-C/EBPϵ), or nuclear extracts prepared from either COS-1 cells overexpressing CHOP (COS/CHOP) or untransfected COS-1 cells (COS/UT) were added to the reactions as indicated. (B) Nuclear-extracted proteins (10 μg) from COS-1 cells transfected with a C/EBPϵ expression vector were incubated with the lactoferrin probe. Unlabeled competitor (cold oligo), C/EBPϵ antibody (anti-C/EBPϵ), or nuclear extracts prepared from COS-1 cells overexpressing CHOP (COS/CHOP) were added to the reactions as indicated. Asterisks indicate the positions of supershifted bands after the addition of C/EBPϵ antibody.
