Figure 4

Incubation with in vitro translated USF1 protein demonstrates that methylation of CpG-4 impedes USF1 binding. EMSAs were performed with 32P-end-labeled probes. In vitro translated USF1 proteins were pre-incubated in the presence of either no competitor or 100-fold molar excess of cold competitor oligonucleotides (C) or consensus sequence (Cs). For supershift assay, 2 μg of the indicated antibody (Ab) were added directly after the addition of the labeled probe. Ab1 and Ab2 are directed against USF1 and USF2, respectively. In methylated probes, dC nucleotide is substituted by an internal methyl deoxyCytidine in CpG-4 dinucleotide.