Firefly renilla dual luciferase assay kit

Luciferase assays were performed to determine the NF-κB activity in response to poly(dA:dT) treatment. The HPV-KER cell line was transfected with the NF-κB reporter construct vector pNF-κB-luc Cis-Reporter Plasmid (Stratagene, La Jolla, CA, USA) and the pGL4.75 [hRluc/CMV] plasmid (Promega, Madison, WI, USA) with the use of the X-tremeGene9 transfection reagent. The treated cells were washed twice with PBS, lysed with passive lysis buffer (Biotium, Hayward, CA, USA) and the luciferase activities in the lysates were measured using the Firefly & Renilla Dual Luciferase Assay Kit (Biotium) and Thermo Luminoskan Ascent (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. All samples were measured three times and the luciferase activity derived from the NF-κB-luc plasmid was normalized to the activity of the Renilla luciferase activity from pGL4.75 [hRluc/CMV] plasmid.

Transient transfection experiments were performed using the X-tremeGENE 9 DNA Transfection Reagent (Roche, Indiana, United States). For the overexpression studies, HPV-KER cells were plated in 12-well plates (100,000 cells/well), transfected for 24 h with 0.5 μg empty pcDNA3.1 vector or pcDNA3.1-TNIP1 vector into which TNIP1 cDNA sequences (OriGene Technologies, Inc., MD, United States) had been inserted. Transfection-grade plasmid was prepared using the E.Z.N.A Endo-free plasmid DNA Maxi Kit (Omega Bio-tek, Inc., GA, United States).
For siRNA-mediated gene silencing, siRNA was delivered by the Santa Cruz siRNA Transfection Reagent (Santa Cruz Biotechnology, Texas, United States) according to the manufacturer's instructions. ON-TARGETplus SMARTpool TNIP1 siRNA or ON-TARGETplus Non-targeting Pool (Dharmacon, Lafayette, United States) constructs were used at a concentration of 10 nM.
A NF-κB luciferase reporter assay was performed using the PathDetect pNF-κB-Luc Cis-Reporter Plasmid (Stratagene, California, USA), pGL4.75[hRluc/CMV] Vector and the Firefly & Renilla Dual Luciferase Assay Kit (Biotium Inc., California, United States), according to the manufacturer's instructions.