Luciferase Reporter Gene Assay

Luciferase reporter assays were performed as described (12 (link)) using a Steady Lite plus reporter gene assay kit (#6066751 Perkin Elmer, Waltham, MA). Briefly, cells were co-transfected with luc reporter constructs, β-gal and test driver constructs in a 96-well plate using Lipofectamine (#18324012) and Plus reagent (#11514015) from Invitrogen. At 24 h post transfection, cell lysates were collected in appropriate lysis buffer (Radio Immuno Precipitation Assay) with protease inhibitors, combined with Steady Lite reagent and analyzed using a Perkin Elmer TopCount plate reader. The signal was normalized to β-gal expression by addition of ortho-Nitrophenyl-β-galactoside substrate and analysis at 430 nm.