Nf κb luc

Manufactured by Promega

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The NF-κB-Luc product is a reporter construct that contains the firefly luciferase gene under the control of an NF-κB response element. It is designed to monitor NF-κB activation in cells.

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NF-κB (10 citations) PGL4.32-NF-κB-LUC (3 citations) PGL-NF-κB-Luc (2 citations)

14 protocols using nf κb luc

Assessing NF-κB Transcriptional Activity

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To assess the NF-κB transcriptional activity, we used the NF-κB–luciferase reporter construct (NF-κB-luc; Promega). HEK293T cells were transiently cotransfected with NF-κB-luc reporter plasmid and phRL-CMV plasmid (Promega) with or without the indicated constructs using the LipoD293 DNA In Vitro Transfection Reagent (SignaGen Laboratories). Cells were lysed and assayed for firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay System (Promega). The data were expressed as the ratio of firefly to Renilla activity.

Matsumoto Y., Dimitriou I.D., La Rose J., Lim M., Camilleri S., Law N., Adissu H.A., Tong J., Moran M.F., Chruscinski A., He F., Asano Y., Katsuyama T., Sada K.E., Wada J, & Rottapel R. (2022). Tankyrase represses autoinflammation through the attenuation of TLR2 signaling. The Journal of Clinical Investigation, 132(7), e140869.

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CRISPR-Cas9 Plasmid Design for Innate Immunity

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For CRISPR–Cas9 plasmids, single-guide RNA (sgRNA) targeting TBK1, IRF3, cyclic GMP-AMP synthase (cGAS), AP-1σ1 and STING were designed using the web tool CRISPOR^{47 (link)}. sgRNAs targeting TBK1, IRF3, cGAS and AP-1σ1 were cloned in a pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (Addgene, 62988), whereas sgRNAs targeting STING were cloned in a pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene, 48138). Both plasmids were gifts from F. Zhang^{48 (link)}. The pEF-Bos-based STING truncations (1–341 and 1–317) and mutations (E360A, LI364/365AA, ELI360/364/365AAA, LR374/375ΑΑ, L364A, L364F and I365A) were obtained by site-directed mutagenesis. pEF-Bos-human TBK1-Flag-His was a gift from S. Cerboni. TBK1(S172A) was generated by single-amino-acid mutation. pCDNA3-HA γ-adaptin 1(AP1G1) (Addgene, 10712) was purchased from Addgene. pCDNA3-HA-AP1S1 was generated by inserting the coding sequences of AP1S1 flanked by 5’ BamHI and 3’ XhoI sites into the pCDNA3 vector. AP1G1(R15E), AP1S1(I103S) and AP1S1(V88D) were obtained by single-amino-acid mutagenesis. The primers used for plasmid constructions and sgRNA sequences are provided in Supplementary Table 1. Plasmids for NF-κB-Luc (Promega, E8491) were purchased from Promega and those for pIFNβ–GLuc were previously described^{8 (link)}. All constructs were confirmed by DNA sequencing.

Liu Y., Xu P., Rivara S., Liu C., Ricci J., Ren X., Hurley J.H, & Ablasser A. (2022). Clathrin-associated AP-1 controls termination of STING signalling. Nature, 610(7933), 761-767.

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NF-κB Luciferase Assay Protocol

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All luciferase assays were performed at 24 h after transfection, using a Dual Luciferase Assay kit (Promega) according to the manufacturer’s instruction. 293T cells were transiently transfected with luciferase reporter construct NF-κB-Luc and pTK Renilla luciferase plasmid (Promega) using PEI, according to the manufacturer’s instruction.

Kim J.A., Choi M.S., Min J.S., Kang I., Oh J., Kim J.C, & Ahn J.K. (2017). HSV-1 ICP27 represses NF-κB activity by regulating Daxx sumoylation. BMB Reports, 50(5), 275-280.

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Investigating EcTRAF4 Regulation of IFN and NF-κB

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To examine the effects of EcTRAF4 on the activity of IFN and the nuclear factor-κB (NF-κB) promoter, luciferase plasmids, including those encoding zebrafish IFN3–Luc, human IFN-stimulated response element (ISRE)–Luc, and NF-κB–Luc (Promega, Madison, WI, USA), were used [37 (link),40 (link)]. In brief, GS cells were co-transfected with 200 ng of plasmid-encoding ISRE–Luc, IFN3–Luc, or NF-κB-Luc and 600 ng of pcDNA3.1-3 × HA or plasmid-encoding HA–EcTRAF4. A total of 50 ng of pRL-SV40 Renilla luciferase vector (Promega, Madison, WI, USA) was used as the internal control. The cells were harvested after 48 h to measure luciferase activities with the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

Wu S., Sun M., Zhang X., Liao J., Liu M., Qin Q, & Wei J. (2021). Grouper TRAF4, a Novel, CP-Interacting Protein That Promotes Red-Spotted Grouper Nervous Necrosis Virus Replication. International Journal of Molecular Sciences, 22(11), 6136.

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Measuring NF-κB Activity in Colon Cancer Cells

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To measure NF-κB transcriptional activity, HCT116 colon cancer cells were transfected with NF-κB-Luc (Promega) reporter plasmids using Polyethylenimine MAX MW 40,000 (PolyScience, Warrington, PA). The transfected cells were cultured for an additional 24 h. Cells stably expressing NF-κB-Luc were treated with hygromycin and cloned. These cells were referred to as HCT116-NF-κB-Luc cells. HCT116-NF-κB-Luc cells were seeded in 96-well plates (2 × 10⁴ cells/well). After a 24-h incubation, the cells were treated with EM and 10 ng/ml TNFα (Perotec, Somerset, NJ) for 24 h. Firefly luciferase activity levels were determined using the Bright GLO Luciferase Assay System (Promega). Basal NF-κB luciferase activity in the control was set as 1.0. Data are expressed as the mean ± SD (n = 4).

Hamoya T., Miyamoto S., Tomono S., Fujii G., Nakanishi R., Komiya M., Tamura S., Fujimoto K., Toshima J., Wakabayashi K, & Mutoh M. (2017). Chemopreventive effects of a low-side-effect antibiotic drug, erythromycin, on mouse intestinal tumors. Journal of Clinical Biochemistry and Nutrition, 60(3), 199-207.

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Luciferase Reporter Assay for Transcriptional Activity

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Luciferase reporter constructs of TNF-α (−1260/+60)-Luc or IL-1α (−1856/+1)-Luc were used in the determination of promoter activity of TNF-α or IL-1α gene,^{23 (link), 24 (link)} and those of NF-κB-Luc, AP1-Luc or IRF3-Luc in the analysis of NF-κB, AP-1 or IRF3 transcriptional activity (Promega). Cells were incubated with Lipofectamine 2000 (Invitrogen) in the presence of each reporter construct and Renilla control vector. Firefly luciferase activity, a reporter, was measured as relative luminescence units and normalized to the Renilla activity, a transfection efficiency.

Park S.H., Kwak J.A., Jung S.H., Ahn B., Cho W.J., Yun C.Y., Na C.S., Hwang B.Y., Hong J.T., Han S.B, & Kim Y. (2017). Piperidylmethyloxychalcone improves immune-mediated acute liver failure via inhibiting TAK1 activity. Experimental & Molecular Medicine, 49(11), e392-.

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NF-κB Luciferase Reporter Assay

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Cells were first stably transfected with a luciferase reporter construct (Nf‐κb‐Luc, Promega). Then, transfected cells were stimulated with IL‐1β (10 ng/mL) for 6 hours. Subsequently, the luciferase assay system (Promega) was used to measure luciferase activity.

Ma C., Wu L., Song L., He Y., Adel Abdo Moqbel S., Yan S., Sheng K., Wu H., Ran J, & Wu L. (2019). The pro‐inflammatory effect of NR4A3 in osteoarthritis. Journal of Cellular and Molecular Medicine, 24(1), 930-940.

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Luciferase Reporter Assay Protocol

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NF-κB-Luc, Myc-Luc, AP1-Luc, and E2F-Luc reporter plasmids (Promega, Madison, WI, USA) were transfected into cells using Lipofectamine 3000 for the luciferase assay. DNA sample was mixed with 100 ng of CMV-Ren plasmid (Promega) as an internal control and co-trnasfected into cells in 6-well plates. Luciferase assay were performed at 48 h after transfection using Luciferase Assay Reagent kit (Promega) and a Synergy NEO (BioTek, Winooski, VT, USA). Values obtained were normalized to Renilla luciferase activity.

Chae U., Lee H., Kim B., Jung H., Kim B.M., Lee A.H., Lee D.S, & Min S.H. (2019). A negative feedback loop between XBP1 and Fbw7 regulates cancer development. Oncogenesis, 8(3), 12.

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Macrophage and HeLa Cell Signaling Assay

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Hela (CLS Cat# 300194/p772_HeLa) and RAW264.7 (CLS Cat# 400319/p462_RAW‐2647) (a murine macrophage cell line) cells were grown in high‐glucose DMEM supplemented with glutamine, penicillin, streptomycin, and 10% FBS (Gibco). pCMV‐GR11 (Addgene, #89105) was a gift from Elizabeth Wilson. pGL4.36[luc2P MMTV Hygro] and NF‐κB‐Luc were purchased from Promega and Beyotime, respectively. Five copies of the AP‐1 promoter were cloned into the BmtI and BglII sites of pNF‐κB‐luc. LPS was from Escherichia coli O55:B5 (Sigma Aldrich; L‐2637). Recombinant human tumor necrosis factor‐alpha (TNF‐α) was purchased from Sangon Biotech. Phorbol myristate acetate (PMA), AZD9567, and DEX were purchased from MedChemExpress (MCE), and all the tested compounds were bought from TargetMol.

Hu X., Pang J., Zhang J., Shen C., Chai X., Wang E., Chen H., Wang X., Duan M., Fu W., Xu L., Kang Y., Li D., Xia H, & Hou T. (2021). Discovery of Novel GR Ligands toward Druggable GR Antagonist Conformations Identified by MD Simulations and Markov State Model Analysis. Advanced Science, 9(3), 2102435.

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HIF and NFkB Promoter Activity Assay

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HIF promoter activity was determined using the pGL4.42 [luc2P/HRE/Hygro] plasmid containing 4 consensus HRE sequences fused to a luciferase reporter (HRE-luc) and NFκB promoter activity was determined using the pGL4.32 [luc2P/NF-κB-RE/Hygro] plasmid containing five consensus NFκB response elements fused to a luciferase reporter (NFκB-luc) (both Promega, Madison, WI). PASMC were co-transfected with 4 μg of plasmid DNA and 0.1 μg pRL-CMV Vector (Promega) on a 10 cm² tissue culture plate at 60% confluence, using Lipofectamine (Gibco BRL) according to the manufacturer's instructions. After 24 h cells were split onto 6-well plates (hypoxia) or BioFlex plates (cyclic stretch) and allowed to adhere. Cells were synchronized in serum-free DMEM for 24 h, returned to DMEM containing 10% serum and exposed to hypoxia or to cyclic stretch as described below. Luciferase activity in protein extracts was determined using the Dual-Luciferase Reporter Assay System (Promega) and a Femtomaster FB12 luminometer (Zylux). Activity was normalized to the internal renilla luciferase control to correct for differences in transfection efficiencies. Where appropriate, cells were treated with 1 μM myxothiazol or 10 μM Helenalin (Calbiochem) prior to luciferase assays.

Wedgwood S., Lakshminrusimha S., Schumacker P.T, & Steinhorn R.H. (2015). Hypoxia inducible factor signaling and experimental persistent pulmonary hypertension of the newborn. Frontiers in Pharmacology, 6, 47.

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