Identification of the putative regulatory elements for MAZ binding within the wild type 34-nt segment. (A) Identification of the MAZ-responsive core sequence. Scanning mutagenesis EMSA was used to define the binding site of the putative regulatory region. 32P-labeled wild type 34-nt segment was used for all experiments in the presence of 200-fold molar excess of unlabeled oligonucleotide competitor as indicated. The sequences of the mutant oligonucleotides m1 to m21 are shown in supplementary Table 2. (B) Summary of the DNA-binding activity of nuclear extract proteins to various mutant competitors to the wild type 34-nt segment. The stars above the sequence shown at the bottom indicate the nucleotides crucial for MAZ binding, and the dark circles below the sequence indicate the nucleotides that are less important for MAZ binding. (C) Western blot analysis of MAZ in nuclear extracts (50 μg protein) isolated from pcDNA3- and pcDNA3/MAZ-transfected HepG2 cells (lanes 1 and 2, respectively). (D) Nuclear extracts were prepared from HepG2 cells and incubated with biotinylated oligonucleotide from the 34-nt region of the edn promoter. The samples were separated by 8% SDS-PAGE and immunoblotted for MAZ. Relative densitometric quantitation of bands in lanes 1–5 are as follows: biotin control, wild type, mutant -62/-48, mutant -74/-65, mutant -62/-48 & -74/-65. The difference between the two groups is statistically significant (P < 0.05), as determined by the Wilcoxon Rank Sum test.