Figure 6. DNA methylation of the 5’ region silences the activity of the gria2 promoter as determined by a transient transfection Luciferase reporter assay.

(A) Physical map of the gria2-Luciferase reporter construct. The 5’ region CpGs in the probe were in-vitro methylated (black lollypop), or mock methylated (empty lollypop). The CpGs in the promoter region were left unmethylated (empty lollypop). Additional constructs were designed as controls; One containing the full promoter but with the 5’ region in a reverse direction (Antisense); second, containing only the 5’ region (5’ region); third containing only the unmethylated promoter (Promoter); forth containing plasmid without any promoter sequence (Empty vector). (B) The indicated constructs were transfected into SH-Sy5y (human neuronal cell line). 48h after transfection the cells were harvested, extracts were prepared and assayed for Luciferase activity and the values were normalized to total protein concentration. Results are average of (n=3) transfections +/- SEM. *p<0.05 **p<0.01 ***p<0.005 determined by a Student t test with Holm-Bonferroni correction.