(A) Schematic of genomic sequence around SNP rs548234. Putative binding sites of various transcription factors are found upstream and downstream of the SNP, and the KLF4-binding site is identified only in risk SNP by using Transfac and oPOSSUM 3.0 programs. (B) KLF binding with the risk allele probe (C/C) not with nonrisk allele probe (T/T) by EMSA. Recombinant KLF4 (left) or nuclear extract (NE) (5 μg) (right) was incubated with either T/T or C/C probe. To identify specificity of binding, either control or anti-KLF4 Ab was added. Arrows indicate KLF4 binding and asterisks indicate supershift. A representative image is shown from 3 independent experiments. (C) In vivo binding of KLF4 to the risk allele. To perform the ChIP assay, MO-DCs from either risk allele carriers (C/C) or nonrisk allele carriers (T/T) were prepared and incubated with either control goat IgG or anti-KLF Ab overnight. After precipitation, qPCR and PCR were performed with a primer set that amplifies the SNP region. The B2R gene was amplified as a positive control. Percentage of input was calculated relative level to the total input. In the box-and-whisker plot, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles (n = 4). The nonparametric, Mann-Whitney test was used for statistics.