Pnfκb luc vector

Manufactured by Takara Bio

Sourced in United States

The PNFκB-Luc vector is a reporter construct that contains the firefly luciferase gene under the control of a promoter element responsive to the transcription factor NF-κB. This vector can be used to monitor NF-κB activity in cells.

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Lab products found in correlation

Luciferase assay, by Promega (2 mentions) β galactosidase enzyme assay, by Promega (2 mentions) Psv β galactosidase β gal control vector, by Promega (2 mentions) Prl tk vector, by Promega (1 mentions) Dharmafect 1, by Thermo Fisher Scientific (1 mentions) Synergy 2 microplate reader, by Promega (1 mentions) Dual glo luciferase assay, by Promega (1 mentions) Fugene 6 reagent, by Roche (1 mentions) Spectramax m3, by Molecular Devices (1 mentions)

5 protocols using pnfκb luc vector

Silencing NF-kB p65 Subunit

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Short interfering RNAs to p65 were purchased from Dharmacon and transfected at 100 nM using Dharmafect 1 (Thermo Fisher Scientific) per manufacturer's protocol at 6 μl of the reagent/well of 6-well plate.
For luciferase assays, MCF7-vector cells were seeded at 100,000 cells/well in 12-well plates and co-transfected with pNFκB-Luc vector (240 ng/well; Clontech, Mountain View, CA) and pRL-TK vector (10 ng/well; Promega, Milwaukee, WI) as a transfection efficiency control using FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). Luciferase activity was measured using Dual Glo luciferase assay (Promega) with a Synergy 2 Microplate Reader.

Shatz M., Shats I., Menendez D, & Resnick M.A. (2015). p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways. Oncotarget, 6(19), 16963-16980.

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Investigating NF-κB Activity Regulation

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K562 cells were nucleofected with the NF-κB luciferase reporter plasmid (pNF-κB-Luc Vector; Clontech) and pFlag-UCKL-1, pControl, or siRNA (siUCKL-1 or sicontrol RNA). Cells were also co-transfected with the pSV-β-galactosidase (β-gal) control vector (Promega) for determination of transfection efficiency. NF-κB activity was assessed using the Luciferase Assay and β-gal activity was assessed using the β-Galactosidase Enzyme Assay, per the manufacturers’ protocol (Promega). Protein concentrations were determined with a BCA protein assay (Pierce). NF-κB readings were normalized to β-gal activity and protein levels in all experiments.

Gullickson G., Ambrose E.C., Hoover R.G, & Kornbluth J. (2016). Uridine Cytidine Kinase Like-1 Enhances Tumor Cell Proliferation and Mediates Protection from Natural Killer-Mediated Killing. International journal of immunology and immunotherapy, 3(1), 10.23937/2378-3672/1410018.

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NF-κB Luciferase Assay Protocol

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The pNF-κB-Luc vector was purchased from Clontech Laboratories (Mountain View, CA, USA). A mixture of 0.02 μg of plasmid and Lipofectamine 2000 at a 1:1 ratio was prepared, added to each well of a 96-well plate (1 × 10⁴ cells/well), and incubated for 36 hr. After replacing the media, ferulate and AAPH were added to the cells for 8 hr. Then the luciferase assay was performed at which point reagents from the ONE-Glo Luciferase Assay System were added to the plate according to the manufacturer’s instructions. Then the luciferase activity was measured using luminometric analysis on a SpectraMax M3 microplate reader (Molecular Devices).

Koh E.M., Lee E.K., Song C.H., Song J., Chung H.Y., Chae C.H, & Jung K.J. (2018). Ferulate, an Active Component of Wheat Germ, Ameliorates Oxidative Stress-Induced PTK/PTP Imbalance and PP2A Inactivation. Toxicological Research, 34(4), 333-341.

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NF-κB Promoter Construct for Luciferase Assay

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A promotor construct of 220 bp was synthesized containing three different binding motifs for the p65 and p50 subunit of NF-κB (5́-GGGGACTTTCC-3́,5́-GGGGATTCCC-3́, 5́-GGGGATTTCC-3́) flanked by restriction enzyme sites for EcoNI (5́) and HindIII (3́) 12,13. An additional NF-κB response element and a TATA-like sequence from pNFκB-Luc vector (Clontech, 631904) were added to the Firefly luciferase gene and introduced into pCDH-CMV-EF1-Puro.

Hassan M.I., Keller M., Hillger M., Binder U., Reuter S., Herold K., Telagathoti A., Dahse H.M., Wicht S., Trinks N., Nietzsche S., Deckert-Gaudig T., Deckert V., Mrowka R., Terpitz U., Peter Saluz H, & Voigt K. (2021). The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes. Computational and Structural Biotechnology Journal, 19, 880-896.

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Investigating NF-κB Activity Regulation

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