2.5. NF-κB Dual Luciferase Reporter Assay

Sample preparation. HEK293T cells were seeded in 12-well plates and transfected 24 h later in triplicates with 0.7 μg of Promega firefly luciferase vector, 0.07 μg of pRL-TK Renilla luciferase vector, plus up to 5 μg of either pBZ empty vector, ORF3a WT or TRAF-mut ORF3a pBZ plasmid per well using calcium phosphate or the polyethylenimine (PEI) transfection method. Media was changed 5 h post-transfection. Six hours before harvesting, positive control cells were treated with 5–10 ng/mL of recombinant human TNF-⍺ (Gibco, Waltham, MA, USA). At 48 h post transfection, cells were harvested using Passive Lysis Buffer (Promega, Madison, WI, USA), sonicated, centrifuged, and stored at −80 °C until the assay was performed.

Assay. Dual-Luciferase Reporter Assay kit (Promega, Madison, WI, USA) reagents were prepared according to the manufacturer’s protocol. A quantity of 15 μL of cell lysate was mixed with 50 μL of Luciferase Assay Reagent II (LAR II) in a 96-well plate, and firefly luciferase activity was measured using a GloMax Discover plate reader (Promega, Madison, WI, USA). A quantity of 50 μL of Stop&Glo reagent was then added, and the Renilla luciferase activity was measured. Results were reported as the ratio of Firefly/Renilla luciferase activities and plotted and analyzed with GraphPad Prism.