Luciferase reporter assay

The LXRE-luciferase vector (TK-LXREx3-Luc vector) was a kind gift from Dr. D. J. Mangelsdorf (University of Texas Southwestern Medical Center). The mouse Arg2 promoter (−1,840 to +245; 2085 bp) was obtained by PCR using mouse genomic DNA as a template and inserted into the luciferase reporter plasmid (pGL3-basic; Promega, Madison, WI). The LXRE (TGGCCTCTAGTAACCA) and STAT1 (TTCCCGGGAA) sites on the Arg2 promoter were mutated to TGGAATCTAGTAATTA and GGTCCGGTTT, respectively (mutated nucleotides are underlined). RAW 294.7 cells were co-transfected with promoter reporters and Renilla luciferase vector (phRL-TK) using X-tremeGENE 9 DNA Transfection Reagent (Roche Applied Science, Penzberg, Upper Bavaria, Germany). After 24 h, firefly and Renilla luciferase activity was measured using a dual-luciferase reporter assay system (Promega) on the Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT).