Transient transfection and reporter gene assay
For luciferase assays, 1 × 106 cells/well were cultured overnight before transfection. Various reporter constructs at equimolar concentrations were transfected using Effectene transfection reagent (Qiagen, Valencia, CA) as described previously (11). After 48 h of transfection, cells were washed twice with phosphate-buffered saline (PBS) and lysed with lysis buffer (Promega). For all the assays, pCH110 (β-galactosidase; Amersham Bioscience Inc.) was also co-transfected and measured to normalize transfection efficiency. The luciferase and β-galactosidase activities were determined according to the manufacturer's instructions (Promega and Tropics, Madison, WI).