Figure 5. Analysis of E2F1, Sp1, KLF15, and E2F4 binding sites by site-directed mutagenesis and the predicted CpG island in the proximal ACSL1 promoter.

(a) Site-directed mutagenesis was carried out in the construct pGL−325/+12. The different constructs were transiently transfected into C2C12 cells. After 48 h, the cells were harvested for the luciferase assay. Results are expressed as the mean ± standard deviation in arbitrary units based on the firefly luciferase activity normalized against the Renilla luciferase activity for triplicate transfections. The error bars denote the standard deviation. The paired Student’s t-test was used to detect significant differences. *P < 0.05 and **P < 0.01. The data shown are representative of two independent experiments. (b) Schematic representation of the proximal promoter region (+1 to −1933 base pairs) of the bovine ACSL1 gene to predict the regions with high GC content. Dashed lines indicate the GC percentage as represented on the y-axis and the x-axis denotes the bp position on the 5′ untranslated region, vertical lines indicate relative positions of CpG dinucleotides. Coordinates are given relative to the translational start site (shown as +1). Arrows indicate the TSS-1, the positions −325 and −141 bp in the promoter.