Luciferase Reporter Assay for Inflammatory Signaling

Luciferase reporter assays were performed as described previously^{34 (link)}. Briefly, 293/TLR4-MD2-CD14 cells (2.5 × 10⁵ cells/ml, InvivoGen, San Diego, CA, USA) were cotransfected with a luciferase reporter plasmid (0.2 μg), pcDNA3.1/His/LacZ (0.1 μg, Invitrogen, Carlsbad, CA, USA) and various expression plasmids (0.25–1.0 μg) in triplicate using TurboFect reagent (Fermentas, Glen Burnie, MD, USA). The murine IL-6 promoter-luciferase reporter (IL-6-Luc (−1277/ + 1)) and the murine TNF-α promoter-luciferase reporter (TNF-α-Luc (−1167/ + 155)) were described previously^{61 (link),62 (link)}; the murine IL-1β promoter-luciferase reporter (IL-1β-Luc (−2045/ + 23)) was generated by PCR-based subcloning into the pGL3-basic vector (Promega, Madison, WI, USA). At 24 h posttransfection, the transfected cells were stimulated with LPS (1 μg/ml) for 24 h. The cells were subjected to a luciferase assay using a luciferase activity assay system (Promega, Madison, WI, USA). All values represent luciferase activities normalized to β-galactosidase activities (Applied Biosystems, Bedford, MA, USA).