Five potential Forkhead box protein A (FOXA, previously known as hepatocyte nuclear factor 3 (Friedman and Kaestner, 2006) binding sites and one HNF-1A binding site (Figure 4) were identified using transcription factor prediction software Promo (Farre et al., 2003; Messeguer et al., 2002). Oligonucleotides were synthesized to cover these potential FOXA binding sites (Table 1c) and tested in EMSA with HepG2 nuclear extract. With all four oligonucleotides, at least two retarded DNA-protein complexes were observed (Figure 5). A non-specific competitor oligonucleotide had no effect on any of the complexes (Figure 5). Competition experiments with unlabeled FOXA consensus oligonucleotide (Smith and Humphries, 2009) disrupted the strong, high molecular weight complex. On the other hand, the complex was intact when a mutated FOXA consensus oligonucleotide was added, confirming that it is a FOXA-specific complex. The FOXA-specific complex was also perturbed in supershift assays with FOXA1 and FOXA2 antibodies, while it was unaffected with control IgG antibody (Figure 5).
