Luciferase assay was performed in ATDC5 cells using an NF-κB Luciferase Reporter (Promega, USA). ATDC5 cells were plated at 25% confluence in 24-well tissue culture plates and NF-κB Luciferase Reporter (50 ng), GPR4 plasmid (500 ng), or vector (pcDNA3.1) were co-transfected with Renilla plasmid (10 ng) on the next day. Twenty-four hours after transfection, the cells were treated with IL-1β (1 ng/ml), with or without 10 μM NE52-QQ57 for 24 h. Then cells were harvested and luciferase activity was assessed with the Dual-Luciferase Reporter Assay System (Promega, USA).
Nf κb luciferase reporter
Manufactured by Promega
Sourced in United States
The NF-κB luciferase reporter is a laboratory tool used to measure the activity of the NF-κB transcription factor. It consists of a reporter gene, the luciferase gene, under the control of an NF-κB responsive promoter. When NF-κB is activated, it binds to the promoter and induces the expression of the luciferase gene, which can then be detected and quantified.
Automatically generated - may contain errors
3 protocols using nf κb luciferase reporter
1
NF-κB Activation Assay in ATDC5 Cells
2
Molecular Cloning for LOXL1-AS1 Regulation
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A fragment expressing LOXL1-AS1 was cloned to the pcDNA3.1(+) vector. The miR-708-5p and miR-101-3p expression plasmids were generated by cloning their precursor sequences to pSuper vector. The short hairpin RNA (shRNAs) targeting LOXL1-AS1 or nonspecific control shRNAs were inserted to the pLKO.1 vector. For generation of the LOXL1-AS1 luciferase reporter, the fragment encoding LOXL1-AS1 was cloned downstream of the pGL3 firefly luciferase coding sequence. To disrupt the potential miR-708-5p target site in LOXL1-AS1, site-directed mutagenesis was performed using a site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA). An NF-κB luciferase reporter was purchased from Promega (Madison, WI, USA).
3
Luciferase Assay for NF-κB Regulation
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The day before transfection, HSCs were inoculated into a 96-well plate at a density of 5 × 103 cells/well. Following the instructions of the Lipo2000 kit, NF-κB luciferase reporter (20 ng) (Promega Corp., Madison, WI, United States) and pRL-TK (5 ng) (Promega Corp., Madison, WI, United States) were co-transfected with shPEG3 or miR-129-5p mimic into HSCs, and the luciferase activity was determined.
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