Luciferase assay analysis of TINF2 promoter truncations defines two significant drops in promoter activity.
A: The name of each TIN2 reporter construct was assigned according to the 5′-end nucleotide numbers of the promoter sequences inserted upstream of the ATG initiation codon. Basic refers to the pGL3-Basic vector. For each transfection, the firefly luciferase activity was normalized to the β-galactosidase activity expressed from a co-transfected β-galactosidase expression vector. The means from three independent experiments are shown for each construct; bars, SD. (***p<0.001). B: Finer promoter mapping of the region between −450 and −351, expressed as in panel A. C: Finer promoter mapping of the region between −148 and −24, expressed as in panel A. D: Verification of the functionality of the TINF2 promoter construct in various cell lines. For each transfection, the firefly luciferase activity was normalized to the Renilla activity expressed from the co-transfected pRL-CMV expression vector and expressed as in panel A. E: Schematic of the core TIN2 promoter construct showing putative Sp1 and NF-κB binding sites predicted using bioinformatic tools. “Drop 1” and “Drop 2” refer to the drops in luciferase reporter activity shown in panel A.