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Figure 1. Sequence features and functional deletion analysis of the human APH-1A gene promoter. A) The 5'-flanking region of the human APH-1A gene was shown. The numbering was based on the transcription initiation site, which was grayed/boldfaced and defined as +1. The potential transcription factor binding sites were underlined/boldfaced. The protein-encoding sequence was italicized; m1 to m6 denote each transcription factor-binding site that was mutated for the mutagenesis study. B) Schematic diagram of the APH-1A promoter deletion constructs consisting of a 5' flanking region with serial deletions cloned into the promoterless vector pGL3-Enhancer. The numbers represent the end-points of each construct. The deletion plasmids were confirmed by sequencing and restriction enzyme digestion, and the digested samples were analyzed on a 1.5% agarose gel. C, D) The constructed plasmids were cotransfected with phRL-SV40 into HeLa cells (C) or rat cortical neurons (D). Luciferase activity was measured after 24 h with a luminometer. Renilla luciferase activity expressed by phRL-SV40 was used to normalize the transfection efficiency. The values represent means ± SE (n
3), *P < 0.05 vs. pGL3-control, **P < 0.001 vs. pGL3-control (by ANOVA with the post hoc Newmann-Keuls test).
