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. 2013 Sep 19;8(9):e76034. doi: 10.1371/journal.pone.0076034

Figure 2. Bioinformatic analysis predicts a putative binding site for thyroid hormone receptors for the ‘A’ variant at rs361525.

Figure 2

Sequences surrounding the -238 A/G polymorphism were analysed by AliBaba 2.1 (http://www.gene-regulation.com/pub/programs/alibaba2/index.html) to predict putative allele-specific transcription factor binding sites (Panel A). The polymorphic nucleotides are capitalised and the binding sites spanning this position are highlighted in bold.

The expression of THRA and THRB in Raw264.7 cells was confirmed by RT-PCR (Panel B). Total RNA was purified from control, LPS stimulated (40 ng/ml, 6 hrs) or LTA stimulated (5 µg/ml, 6 hrs) cells, reverse transcribed and expression was detected by gene specific PCR primers (THRA: 480 bp product, THRB: 430 bp product).

EMSA was performed to demonstrate binding of TR to the “A” variant of rs361525 (Panel C). Specificity of binding was ascertained by competition with a 100-fold molar excess of cold DNA fragments added to the reaction mixture (lane 3). For super-shift assay, the nuclear extract were incubated with an anti-TRβ polyclonal antibody.