Fig. 4

EMSA confirms allele-dependent binding of CEBPB at rs7889204. Panel A visually depicts the computational prediction that the rs7889204 T allele creates a stronger binding site for CCAAT Enhancer Binding Protein Beta (CEBPB). The sequence logo of the CEBPB binding motif (below) shows the DNA-binding preferences of CEBPB. Tall nucleotides above the dashed line indicate DNA bases that are preferred by CEBPB, whereas bases below the dashed line are disfavored. The y-axis indicates the relative free energies of binding for each nucleotide at each position. The height of each nucleotide can be interpreted as the free energy difference from the average (ΔΔG) in units of gas constant (R) and temperature (T). The sequence located in the AGTR2 locus is shown directly below the x-axis, with the T allele for rs7889204 at the bottom. The T allele changes the sequence from C (most disfavored) to T (most preferred). Panel B shows the experimental validation of allele-dependent binding of CEBPB to rs7889204 using an electrophoretic mobility shift assay (EMSA). Arrows indicate allele-dependent binding of CEBPB (bottom arrow) and a “super shift” of the protein-DNA complex induced by the binding of DDK tagged antibody (CEBPB tagged with DDK motif) to the complex (top arrow)