DNA binding affinity of C/EBPϵ is altered by phosphorylation. Gel shift analysis was carried out on nuclear extracts derived from Jurkat cells transfected with wild-type and mutant C/EBPϵ expression plasmids and an oligonucleotide containing the C/EBP site from the mim promoter. (A) Nuclear extracts from transfected Jurkat cells give a slow migrating band with this oligonucleotide. This retarded band can be competed by 100-fold excess of cold double-stranded (ds) oligonucleotide and supershifted by C/EBPϵ-specific antisera. (B) EMSA was carried out using the same amount of nuclear extract with decreasing concentrations of hot oligonucleotides. The gel shifts were analyzed on a Phosphoimager (Molecular Dynamics, Piscataway, NJ) to obtain the radioactive counts of bound and free probe. Scatchard analysis determined the affinity of these C/EBPϵ proteins for the C/EBP site in the mim promoter as follows: C/EBPϵ 7.69 nM; C/EBPϵT75D 0.48 nM.