Figure 2.

Inhibition of ATF2/c-Jun and CREB binding by the −53 CpG methylation. (A) EMSA using probes corresponding to the proximal AP1 site of the IFN-γ promoter methylated at the indicated CpGs and nuclear extracts from the AE7 (T_H1) cells. (B) EMSA in the presence of competitor oligonucleotides. The assays were carried out with AE7 nuclear extract and labeled, unmethylated AP1 probe in the presence of increasing amount of unlabeled, methylated or unmethylated, or nonspecific (NFAT) competitor oligonucleotides. The first lane was from the incubation without competitor oligonucleotide. The remaining lanes were from incubations with the indicated unlabeled competitor oligonucleotides. (C) EMSA in the presence of specific antibodies. The assays were carried out with nuclear extracts from either AE7 or D10 (T_H2) cells and labeled, unmethylated AP1 probe in the absence (No Ab) or presence of the indicated antibodies. The EMSA with labeled, methylated AP1 probe serves as controls. The anti-ATF1 antibody crossreacts with CREB. Data shown are representative from one of the three to five experiments.