HNF-1α binding affinity in wild-type and mutant human P2 promoter oligonucleotides. (a) Schematic illustration of oligonucleotides containing the human HNF-4α P2 promoter HNF1 site (bold), and sites containing either the –181G→A mutation (P2 HNF1G→Α) or an artificial mutation intended to completely disrupt HNF1 binding (P2 HNF1→SAC). Mutated bases are in lower case. (b) EMSA of radiolabeled wild-type HNF1 probe. Lanes 1 and 2: incubation with translation reactions using empty vector and pCMVTag-HNF1α, respectively. Lane 2 represents the maximal binding obtained in the absence of any cold competitor. Lanes 3–11: same as 2, except for preincubation with the indicated unlabeled probes at 1×, 10×, and 100× excess relative to the labeled probe. Lanes 12 and 13: same as 2, except for preincubation with either anti–HNF-1α or preimmune antisera, respectively. Similar results were obtained with mouse pancreatic nuclear extracts (not shown). (c) Results from two experiments such as the one shown in b were used to calculate oligonucleotide concentrations required for half-maximal displacement (HNF1G→A, 9.67 ± 1.45 nM; HNF1, 1.36 ± 0.22 nM). *P < 0.05; **P < 0.01.