FIGURE 5.

EMSA of binding to the CHRNA7 promoter AP-2 binding site at −71. Double-stranded oligonucleotides containing the normal (wt) or the mutated (mt) AP-2 site and the flanking sequences as found in the human most common sequence CHRNA7 promoter were 3′-end-labeled with biotin and incubated with nuclear extracts prepared from SH-SY5Y, SK-N-BE, SK-N-MC, HeLa (not shown), HepG2 (not shown), or SH-EP1 (not shown) cell lines as indicated. Extract was present in lanes 2–6 of each gel, wild type labeled probe was used in lanes 2–4 and 6, and mutated labeled probe was used in lane 5. Unlabeled competitor wild type probe was present in 100-fold excess in lane 3, and unlabeled competitor mutated probe was present in 100-fold excess in lane 4. Anti-AP-2α antibody was used for supershifting in lane 6. Protein-DNA complexes are indicated by the arrows labeled AP-2α, and the antibody supershifted complexes are indicated by the arrows labeled SS. Protein complexes indicated by the arrows labeled with asterisks are unknown ternary complexes. EMSAs were replicated two times for each cell extract, and three separately prepared extracts were used for each cell line.