Transcriptional activity of rs12041331 and enhancing role of methylation at the G allele. EMSA analysis to characterize the methylated G allele vs the G allele and A allele binding properties to nuclear proteins. EMSA were performed using 5′biotinylated probes corresponding to location chr1:156869698-156869731 (GRCh38/hg38 Assembly) and including rs12041331. (A,C) Biotinylated double-stranded probes containing the G allele with (“Gmeth”) or without (“G”) methylation, or the A allele (“A”), were incubated in the presence of a 10× amount of cold probe (lanes 1-3), or alone with a fixed amount of nuclear extract from CHRF (A) or MEG01, EA.hy926, or HEK-293 cells (C). Lanes 7-9 show unbound biotinylated probes in all cases. Methylation significantly enhanced binding of the probe to nuclear extract proteins as shown by the increased normalized intensity of the signal in lane 4 vs that in lane 5 and 6 (B), quantified for 3 replicates of CHRF cells (A, mean ± standard deviation) and for the other 3 cell lines investigated (C), expressed as lane Integrated Density. (D) PROMO-based putative binding sites search for CpG-SNP region (25 bp). The G allele (top panel) produced specific binding prediction for c-Jun (indicated as number 7) and ATF3 (indicated as number 8), whereas for the A allele (bottom panel), YY1 (indicated as number 3) is predicted to bind the SNP site.