(A) Luciferase expression from constructs containing the risk versus non-risk variants of SNPs 2, 4, and 5 was measured in SHSY5Y, SK-N-MC, and HEK293T cells. White and red circles represent the non-risk and risk variants, respectively (see Figure 2 for additional features of the depicted constructs). All assays were performed in quadruplicate and repeated at least three times. Error bars represent the standard error of the mean. The vertical dashed line represents the RLA measured for the construct containing the non-risk haplotype (set at 1.0 RLA); note that this reflects a different scale than the RLA depicted in Figure 2. For all three cell lines, only the construct containing the SNP 2 risk variant (denoted with a red arrow) yielded a significant RLA difference compared to the construct containing the non-risk haplotype, as analyzed using an unpaired two-sided t-test (P = 8.32×10−10, SHSY5Y; P = 3.92×10−7, SK-N-MC; P = 4.02×10−8, HEK293T). (B) EMSA testing the binding of SHSY5Y nuclear protein(s) to probes containing the SNP 2 risk versus non-risk variant. The presence of a competitor is denoted above each lane: -, no competitor; R, risk competitor; N, non-risk competitor; AP2, competitor containing an AP2-binding site (negative control); and *, 10-fold and **, 100-fold excess of competitor, respectively. (C) EMSA testing the binding of SHSY5Y nuclear protein(s) to probes containing the SNP 2 risk variant in the presence of competitors containing binding sites for CRX, OCT-1, and AP2 (negative control). (D) Supershift EMSA testing the binding of SHSY5Y nuclear protein(s) to probes containing the SNP 2 risk variant in the presence of anti-CRX or -OCT-1 antibody or general rabbit antiserum.