Fig. 2: Preliminary identification of the functional binding sites in the promoters of the non-receptor genes APN5, APN6, and ABCC1 by a dual-luciferase reporter assay.

The FTZ-F1 expression vector was co-transfected with various truncated promoters of non-receptor genes APN5 (a), APN6 (b), and ABCC1 (c). The results are presented using the same fish-like structure as Fig. 1. The lower “bones” represent the second set of deletions created within the region identified as containing the functional binding site from the initial set of deletions. The horizontal numbers represent the nucleotide position of the different truncations relative to the start codon. d–f The effect of FTZ-F1 on wild-type or mutated promoters of APN5, APN6, and ABCC1 genes. A series of recombinants comprising 5–6 base mutations in each of the promoter regions was constructed and co-transfected with an FTZ-F1 vector to precisely identify the position of the functional FBSs. An empty pAc5.1 vector was used as a control (a–f). The relative luciferase activity (fold) was calculated based on the value of the control, which was assigned an arbitrary value of 1. Data were presented as mean values (a–c) and mean values ± SEM (n = 3) (d–f), ns, not significant, p values are shown. Differences between wild-type and mutated promoters were tested by one-way ANOVA with Tukey’s test (d–f). Source data are provided as a Source Data file.