Fig 1: Allele-specific transcription factor binding at the genomic region surrounding rs7173049.(A)Transcription factor binding sites overlapping SNP rs7173049 (marked in red) regions (±15 bp) as predicted by the PROMO bioinformatics program. Differential binding to sequences containing the protective G allele (rs717-G) compared to sequences containing the A allele (rs717-A) was only predicted for T3R-beta1 (THRß1). Each gray/orange box indicates one transcription factor: GR-alpha, ER-alpha, XBP-1 and YY1. (B) Supershift assays with 31 bp biotinylated DNA probes containing rs7173049 G allele (rs717-G), nuclear extracts from hTCFs and specific antibodies against candidate transcription factors RXRa-1,2,3, beta (RXRß) and gamma (RXR?), RARa/ß/? and thyroid hormone receptor alpha/beta (THRa/ß) showing disruption of the shifted DNA–protein complex (solid arrow) and formation of supershifted bands (dotted arrows) with the THRa/ßantibody. Dotted black line indicates a cut and junction of two parts of the same membrane. (C) Supershift assays with THRa/ß antibody attenuated the shifted DNA–protein complex (solid arrow) and produced distinct supershifted bands (dotted arrows) with DNA probes containing the protective allele G of rs7173049 (rs717-G). No supershifted bands were detected with probes containing the A allele (rs717-A). Bands which were not competitively inhibited by unlabeled oligonucleotides (arrowhead) were considered unspecific. (D) Increasing amounts of recombinant THRß protein were used in positive control experiments to show a specific interaction with the DNA probe containing the protective G allele of rs7173049 (rs717-G). DNA–protein complexes (arrows) presumably represent thyroid hormone receptor (THR) monomers and homodimers. (E) Real-time PCR analysis of THRB, CRBP1, CRABP2, RARA and RXRA mRNA in hTCFs (n = 4) transfected with THRB-specific siRNA or scrambled control siRNA; expression levels were normalized relative to GAPDH and expressed relative to mock-transfected controls (dashed line; (*P < 0.05; **P < 0.01 ***P < 0.001).