Dual Luciferase Assay for NFκB Activation

Dual luciferase reporter assays were performed as previously described21 (link)24 (link). Keratinocytes were seeded at 3 × 10⁵ cells/mL in 24 well dishes the day prior to transfection. Cells were transfected with NFκB-driven firefly luciferase reporter, Renilla luciferase (RLTK) expressing plasmid as transfection control, and each of the plasmids encoding HPV proteins using the PEI reagent (PolySciences). At 24 hours post transfection cells were stimulated with the appropriate reagents for 6–24 hours (see figure legends for details – all ligands purchased from Invivogen) prior to lysis in passive lysis buffer (Promega). Firefly luciferase levels were normalised to Renilla levels and fold induction values were calculated relative to the normalised activity of the mock.