Luciferase reporter assay

NIH3T3 cells were grown in Dulbecco's minimal essential medium (DMEM) containing 10% calf serum. Triplicate samples of 1x105 cells in 35mm plates were transfected using lipofectin (Gibco BRL) with 1 mg of the reporter plasmid, 0.05 to 5 mg of various expression vectors, and 1 mg of pCMV-b-gal. pcDNA3 plasmid DNA was added to the transfections as needed to achieve the same total amount of plasmid DNA per transfection. Six hours after transfection, the cells were fed with fresh medium (DMEM with 10% calf serum) and incubated overnight. The cells were then serum-starved for 24 hours in DMEM containing 0.5% calf serum. Cell extracts were then prepared and luciferase assays were done using the Luciferase Assay System (Promega). Luciferase activities were normalized with respect to parallel b-gal activities, to correct for differences in transfection efficiency. b-gal assays were performed using the b-Galactosidase Enzyme Assay System (Promega).