Electrophoretic mobility shift assay showing inhibition of binding by DNA methylation. (A) Unmethylated wild-type probe (WT) from –68 bp through –42 bp relative to the transcription start site containing 2 CpGs formed 3 specific DNA-protein complexes with nuclear extract from HaCaT cells (c1-c3 as indicated by white arrows, lane 1) that could be specifically competed for by the excess from high to low (× 200 and × 20) of the unlabeled WT ds-oligonucleotide (lanes 2-3), but less by the excess (× 200 and × 20) of the unlabeled methylated (Me) probe (lanes 4-5). Methylation of the 2 CpGs (lane 6) in the probe sequence affected the formation of the DNA-protein complexes. (B) Unmethylated wild-type probe also formed specific DNA-protein complexes with nuclear extract from nonexpressing Namalwa cells (lane 1) that could be specifically competed for by the excess from low to high (× 20 and × 200) of the unlabeled WT probe (lanes 2-3). The upper complex, which runs with the same mobility as c1 formed with HaCaT nuclear extracts, was not or to a lesser extent competed for by the excess (× 20 and × 200) of the unlabeled but methylated (Me) probe (lanes 4-5). Binding to the methylated probe (lane 6) abrogated the formation of DNA-protein complex c1. (C) The binding activities were competed for with excess from high to low (× 200 and × 20) unlabeled Sp1 probe (lanes 2-3). As a control, the labeled Sp1 oligo was used to show that by competition with excess unlabeled oligo, the specific Sp1 complex (S) was competed away (lanes 5-6).