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. 2015 Aug 17;5:13120. doi: 10.1038/srep13120

Figure 3. The WNT5A −5244 CC allele attenuates its binding affinity to SOX9.

Figure 3

(A) MatInspector identified that the binding site of SOX9 is abolished by a −5244 GCCA to CC variation. The percentage matrix similarity score (in parentheses) is 0.941 (100% match to the matrix yields a maximum score of 1.00). Basepairs in capital characters indicate the core sequence. Basepairs in red characters show that the matrix exhibits a high conservation (Ci value > 60) at this position. (B) A competitive electrophoretic mobility shift assay on −5244GCCA/CC was performed using HEK293 nuclear extract. Lanes 1-7: biotin-labelled GCCA probe plus HEK293 nuclear proteins; Lanes 8-14: biotin-labeled CC probe plus HEK293 nuclear proteins. In the presence of nuclear extract, protein-DNA complexes are observed (lanes 2 and 9). 5- and 50-fold excesses of the unlabelled probes were used. The protein-DNA complexes were competed by increasing amounts of the unlabelled GCCA (lanes 3 and 4, and lanes 10 and 11) and CC probes (lanes 5 and 6, and lanes 12 and 13). The GCCA probes had a higher binding activity than the CC probes. (C) ChIP assays with HEK293 and a human tissue sample from right atrial. The presence of the SOX9-binding WNT5A enhancer was verified by PCR.