Luciferase-based IFN-β promoter activity assay

HEK293T cells were grown to 60% confluence and transfected with a combination of expression constructs encoding an IFN-β inducible firefly luciferase (5 ng), constitutively expressed Renilla luciferase (5 ng), RIG-I_(2CARD) (25 ng) and various PLP2-V5 constructs (50 ng). The total amount of DNA was kept constant by the addition of an empty pcDNA3.1 vector. Transfections were performed as described before using the calcium phosphate transfection method. At 18 h.p.t., cells were lysed in passive lysis buffer (Promega) for 30 minutes. Cell debris was separated from the lysate by centrifugation cell lysate supernatant luminescence was measured with the dual-luciferase reporter assay system (Promega) using the EnVision Multilabel plate reader (Perkin Elmer). Firefly luciferase activity was normalized to Renilla luciferase and statistical significance was determined using an unpaired two-tailed Student’s t-test.