Figure 3.

rs11708067 exhibits allelic differences in transcriptional activity and protein binding in 832/13 rat insulinoma cells. A: The 231-bp segments containing allele A or G of rs11708067 were cloned into a pGL4.23 luciferase reporter vector upstream of the minimal promoter in both orientations (forward and reverse). The relative luciferase activities of plasmids transfected into 832/13 cells normalized to an empty vector control are shown on the y-axis. Error bars represent SD of three to five independent clones per allele (t tests). Black triangles, empty vector; black circles, rs11708067-A allele; white circles, rs11708067-G allele. B: EMSAs with biotin-labeled probes containing either the A or G allele of rs11708067. Probes were incubated with 2.2 µg 832/13 rat insulinoma nuclear lysate. The arrows indicate differential protein binding to the A allele, which is competed away by 240-fold excess competitor DNA containing the A allele (lane 3). EMSAs were performed six separate times with consistent results. Antibodies to CEBPB and NKX2.2 (8 µg) were tested for supershifts in lanes 4 and 5, respectively, and lanes 9 and 10, respectively.