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. 2006 Jan 27;394(Pt 1):173–183. doi: 10.1042/BJ20051296

Figure 8. Luciferase reporter assay determined the site on the 1B promoter region responsible for regulation of the isoform after RA removal.

Figure 8

(A) Diagrammatic representation of 5′-isoforms of DMT1. The Figure shows two alternative start sites of transcription (1A and 1B) with their respective 5′-flanking regions (indicated ‘Pr’). The analysis of that region showed probable binding sites for NF-κB and Sp1. (B) Luciferase reporter assay showing decrease in relative luciferase activity a day after removal of RA when WT promoter (WT-day 4 versus WT-day 5) and 3′-end of the promoter [Prom (−504)-day 4 versus Prom (−504)-day 5] was used. The results are shown as relative luciferase activity (RLU) normalized to β-galactosidase (b-gal) activity. Results are means±S.D. for three separate experiments. A graphical representation of the transfected plasmid is shown. (C) Luciferase reporter assay showing lack of RA removal response when promoter with NF-κB mutation (NFm-day 4 versus NFm-day 5) was used. The response was preserved when promoter with Sp1 mutation (Sp1m-day 4 versus Sp1m-day 5) was used. As a control, luciferase activity was measured in cells transfected with pGL3-control plasmid, which showed no change after RA removal (pGL3c-day 4 versus pGL3-day 5). The results are shown as relative luciferase activity (RLU) normalized to β-galactosidase (b-gal) activity. Results are means±S.D. for three separate experiments. A graphical representation of the transfected plasmid is shown.