Fig. 5.

Luciferase and EMSA analyses of variants assessing regulatory potential. The regulatory potential of intronic SNPs cfa25:45,444,053 (A) and cfa25:45,445,768 (B) was assessed by luciferase reporter assay in the Jurkat T-cell line. After transfection, cells were left unstimulated or stimulated with PMA and ionomycin for 12 h. The risk alleles for both SNPs correlate with lower luciferase levels in nonstimulated and stimulated cells. RFU, relative fluorescence units. Bars represent mean values ± SEM. Statistical analysis was done using an unpaired t test. An EMSA was performed to test for differential DNA binding of Jurkat-cell nuclear extract between the nonrisk and risk alleles at cfa25:45,444,053 (C) and cfa25:45,445,768 (D). (C) Binding to the risk allele at cfa25:45,444,053 appears stronger than the corresponding binding to the nonrisk allele at two locations (red arrowheads), and binding of one transcription factor may be lost in the risk allele (blue arrowhead). (D) At cfa25:45,445,768, binding to the risk allele at one location appears stronger than in the nonrisk allele (red arrowhead). Lab. P., labeled probe; NR, nonrisk allele; Nuc. Ex., Jurkat nuclear extract; R, risk allele; Unlab. P., unlabeled probe.