Pnf κb luc

Stable cell lines (CMV-TRAF6 cells or TRAF6-sh2 cells) in 24-well plates were transfected with 500 ng pNF-κB-luc and 50 ng pRL-TK Renilla luciferase (Promega, USA), the pRL-TK plasmid as a control for transfection efficiency. At 12 hpt, cells were mock-infected, CSFV-infected, or ligand-stimulated for an additional 12 h. The activities of Firefly and Renilla luciferase were determined using the dual-luciferase reporter assay system (Promega) according to manufacturer instructions. Assays were conducted the independent experiments at least in triplicate. The data represent relative Firefly luciferase activity normalized to Renilla luciferase activity.

Huh7.5 cells were cotransfected with the plasmid pNF-κB-Luc (Promega), expressing firefly luciferase, and the pRL-SV40 vector (Promega), expressing Renilla luciferase, in a 10:1 mass ratio using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h of transfection, the cells were pretreated with different inhibitors for 12 h and were then treated with or without 200 μM of H₂O₂ for another 2 h. The luciferase activity of the cell lysate was determined with a dual-luciferase reporter assay kit (Beyotime Biotechnology) according to the manufacturer’s protocol. The relative luciferase activities were calculated by the ratio of the intensity of firefly luminescence to the intensity of the reference Renilla luminescence.

HEK293T cells were seeded in a 12-well plate (Thermo Scientific) 24 h before transfection. Cells were transfected with the indicated amount of pcDNA-BCL10, or pcDNA-BCL10–LinUBL73P-4X as well as with 250 ng of pNF-κB Luc (Promega) and 25 ng of pRL-TK Renilla (Promega) for normalizing transfection efficiency. Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction, and 24 h after transfection, luciferase activity was assayed by Dual-Glo® Luciferase Assay System (Promega).

PK15 cells were cultured in 24-well plates and cotransfected with 100 ng of the luciferase reporter pNF-κB-luc and 10 ng of the constitutive Renilla luciferase reporter pRL-TK (Promega). After transfection for 24 h, the cells were infected with PEDV or BVDV or coinfected with both at an MOI of 1. Then, 24 h later, the cells were lysed and subjected to luciferase assays using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. The results are shown as the means ± SD of triplicate wells and expressed as relative luminescence units (RLUs).

Cells were transfected with pNF-κB-Luc and SV-40-Renilla-Luc (Promega, Madison, WI). At 20 h post transfection, the cells were serum-starved for 16 h, pretreated with EGCG for 1 h, and then treated with IL-1beta (20 ng/ml). The cells were then collected in passive lysis buffer. The cell lysates were analyzed for their firefly and Renilla luciferase activities using a dual luciferase assay kit (Promega).

RAW264.7 cells (2 × 10⁵ cells) were transfected with 0.2 μg of pNF-κB-Luc (Promega, Madison, WI, USA) and 0.2 μg of pRL-TK (Promega) using the Neon® Transfection System (Life Technologies). After 36 h, transfected cells were treated with coffee extract or pyrocatechol for 1 h prior to the stimulation with LPS (1 μg/mL) for 6 h. Luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega)^{42 (link)}.

For reporter gene assays, A549-NF-κB-LUC cells (see “Cells, viruses, and reagents” above) were seeded in 96-well plates 24 h prior to infection. Cells were lysed in passive lysis buffer (Promega), and the firefly luciferase activity was measured using a FLUOstar luminometer (BMG). The fold induction of NF-κB reporter activity was calculated by normalizing each result to the luciferase activity of the nonstimulated mock-infected control cells. For conventional reporter gene assays in HEK293T cells, cells were seeded in 96-well plates and transfected with 60 ng pNF-κB-LUC (R. Hofmeister, University of Regensburg, Regensburg, Germany) and 10 ng pTK-Ren (Promega) using polyethylenimine (PEI; Sigma-Aldrich), according to the manufacturer's protocol, 24 h prior to infection with the vv811 recombinant viruses. Cells were lysed, and firefly and renilla luciferase activities were measured as described above. In each case, the firefly luciferase activity was normalized to the renilla luciferase activity and the fold induction was calculated by normalizing each result to that for the nonstimulated mock-infected control cells. Experiments were performed in quadruplicate and repeated at least 3 times.