Quantifying Promoter Activity via Luciferase Assay

TAP1, TAP2, TAPBP and PSMB9 promoter sequences were amplified from genomic DNA and then cloned into the pGl3 luciferase (luc) vector (Promega, Fitchburg, USA) as recently described [20 (link), 48 (link)]. For transient transfections, 1 × 10⁵ cells were cultured in 100 µl OptiMEM (Invitrogen), followed by transfection with 0.3 µg promoter constructs and 0.016 µg β-galactosidase (β-gal) vector using Lipofectamine 2000 (Invitrogen, Waltham, USA) as transfection reagent according to the manufacturer’s instructions. 48 h after transfection, the luc activity was determined by adding the luc substrate (Promega) using a luminometer and normalized to the transfection efficiency determined by ß-gal enzyme activity. Experiments were independently done three times using triplicates.