The regulation of the APP promoter was probed in cultured neurons using the Dual-Luciferase Reporter Assay System (Promega) that allows sequential measurements in a single sample of luminescent signals of firefly luciferase transcribed from the APP reporter and Renilla luciferase transcribed from a control promoter. We inserted the wild-type human APP promoter region (from −554 to +147; PCR-amplified from human genomic DNA extracted from HEK293T cells) into the KpnI and BglII sites of the promoter-less firefly luciferase vector pGL3-Basic (Promega), and obtained a matching mutant APP promoter construct with an AP-1 site substitution (changing the TGATTC sequence to TAATTA) by site-directed mutagenesis. The wild-type or mutant APP reporter plasmids (500 ng) were introduced at D9 together with a control plasmid (pRL-TK; Promega) encoding Renilla luciferase transcribed from a the herpes simplex virus thymidine kinase (HSV-TK) promoter (50 ng) into cultured human neurons in 24 well plates by liposome transfection (Lipofectamin 2000, Life Technologies). The co-transfection of the control plasmid which constitutively expresses Renilla luciferase with the test plasmid enables normalization of the transfection efficiency and recovery during the cellular extraction for each sample in the reporter assays. Each reporter experiment included extracts from sham-transfected cells as a reference control.
