Figure 2.

FZD6 rs138557689/C creates an allele-specific protein-binding complex and decreases FZD6 promoter activity. (A) Electrophoretic mobility shift assays (EMSA) were performed using nuclear extract from Cos7 cells. Samples were incubated with P³²-labeled oligonucleotides containing either the ancestral A or alternate C alleles, or with unlabeled ancestral A or alternate C serving as specific competitors. Poly-dCdG was used as a nonspecific competitor. Negative controls were run using labeled probes without the nuclear extract. Bands were observed with the C allele only and the alternate band was competed out with C competitor only. (B) Hek293T cells (100,000 cells/well) were seeded for 24 h and cotransfected with ancestral or alternate promoter construct and Renilla reporter construct. Luciferase activities were normalized to the internal Renilla control. Data represent mean values ± SD from three independent experiments. Alternate C allele showed significant decrease in activity (*P < 0.001, unpaired t-test).