Prl tk renilla luciferase based reporter control vector

HEK293T cells were transiently transfected using the calcium phosphate method, as described previously^{42 ,45} , with 5 μg of pGL3 firefly luciferase-based reporter vector and 1 μg of pRL-TK Renilla luciferase-based reporter control vector (Promega, Madison, WI). Forty-eight hours following transfection, the cells were assayed for both firefly and Renilla luciferase activities using the manufacturer's suggested protocol for the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Briefly, cells were harvested, washed in PBS pH 7.4, and lysed using the manufacturer's “passive lysis” buffer. Using automatic injectors within a Flexstation-3 (Molecular Devices, Sunnyvale, CA), firefly luciferase substrate was added to the cellular lysate and luminescence was recorded in a white, clear-bottom 96-well plate for 10 seconds. Firefly luminescence was then quenched and Renilla luciferase substrate was added to the lysate and luminescence was recorded for 10 seconds. Data were normalized to luminescence induced by Renilla luciferase activity and reported as fold-change from pGL3-M transfected HEK 293T cells. All data are representative of biologic triplicates.