Figure 5

CpG methylation blocked the binding of KLF6 to TFPI-2 promoter. (A) CpG methylation in core matrix decreased the binding ability of KLF6 in vitro. Competition were used to determine the affinity of K6, Kc, methylated K6 (mK6) and muted K6 (Kmute). A 50- fold, 100-fold and 200-fold (50×, 100×, 200×) molar excess of cold K6, Kc, mK6 and Kmute cold probe were chased with labeled Kc. All the sequences were listed in table 1 (B) ChIP reveals that hypermethylation blocks KLF6 binding to the TFPI-2 promoter in vivo. Both CpG Unmethylated MCF-7 and methylated MDA-MB-435 cells were tested for binding of KLF6 by ChIP in vivo. Templates for PCR corresponded to the input used in the ChIP assay (input) and DNA obtained from immunoprecipitations performed in the absence (No antibody) or presence of anti KLF6-specific antibody.