|
Fig. 7
Genomic SCN1A fragment harbouring SNP rs7587026 has promoter activity. a DNA sequence and schematic representation of genomic fragments of SCN1A cloned in luciferase constructs. The 50 bp fragments are denoted as 50 bp-rs7587026-G (WT) (for the major allele/wild type sequence), 50 bp-rs7587026-T for the minor allele sequence and as 50 bp-Scramble3 (for the scramble sequence); the 20 bp fragments are denoted as 20 bp-rs7587026-G (WT) (for the major allele/wild type sequence) and 20 bp-rs7587026-T for the minor allele sequence. b Luciferase activity of NS20Y cells transfected with the 50 bp luciferase fragments with both genotypes and a scrambled sequence (constructs: SCN1A-50 bp-rs7587026-G(WT)-Luciferase, SCN1A-50 bp-rs7587026-T-Luciferase or SCN1A-50 bp-Scramble3-Luciferase) and 20 bp luciferase fragments of both genotypes (constructs: SCN1A-20 bp-rs7587026-G(WT)-Luciferase or SCN1A-20 bp-rs7587026-T-Luciferase). Luciferase values were normalized to Renilla values. Data is represented as mean ± SEM (one-way ANOVA, Tukey’s multiple comparison test, n = 4). c Luciferase activity of NS20Y cells co-transfected with the 50 bp-SCN1A fragments (SCN1A-50 bp-rs7587026-G(WT)-Luciferase or SCN1A-50 bp-rs7587026-T-Luciferase) and pCMV-Sox2-T2A-GFP (50 ng). Luciferase values were normalized to the untreated condition (data is represented as mean ± SEM, n = 4, One sample t test). d,e EMSA performed with HEK293T cells overexpressing pCMV-Sox2-T2A-GFP or hypB-CAG-2A-eGFP (control) with the 50 bp SCN1A fragments (d) and the 20 bp SCN1A fragment (e). Scr denotes scrambled sequences (sequences in Supplementary Table 2, online resource). f EMSA reaction with nuclear protein extracts from mouse brain extract and the 50 bp SCN1A fragments. Numbers denote DNA–protein complexes