pAECs grown at 80% confluence in six-well plates were co-transfected with 500 ng of pNF-κB-Firefly Luciferase plasmid (Agilent) expressing luciferase under control of NF-κB responsive elements and 50 ng of pRL-TK-Renilla-Luciferase expression vector (Promega, Charbonnières-les-Bains, France), which allowed the normalization of the transfection efficiency. Transfections were performed using 3.75 µL of Lipofectamine 3000 (Fisher Scientific, Illkirch-Graffenstaden, France) and 2 μL of reagent P3000 (Fisher Scientific, Illkirch-Graffenstaden, France) per well. Cells were treated with DMSO or CSC (100 µg/mL), whether combined or not with RAP (12.7 µg/mL), 24 h after the transfection for 48 h. Firefly and Renilla luciferase activities were then measured with a FB12 luminometer (Berthold, Thoiry, France) in the cellular extracts using the dual-luciferase reporter assay system (Promega, Charbonnières-les-Bains, France), according to the manufacturer’s instructions. This experiment was repeated three times (each condition in duplicate).
Renilla luciferase expression vector prl tk
Manufactured by Promega
Sourced in France, United States
The Renilla luciferase expression vector pRL-TK is a plasmid that expresses the Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase (HSV-TK) promoter. It is used as a reporter gene to measure gene expression in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Fb12 luminometer, by Berthold Technologies (1 mentions)
Dual luciferase reporter dlr assay system, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Ptal luc vector, by Takara Bio (1 mentions)
Lipofectamine 3000 reagent, by Promega (1 mentions)
7 protocols using renilla luciferase expression vector prl tk
1
NF-κB Transcriptional Activity Assay
2
Myd88 Overexpression Modulates NF-κB Activity
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A20 cells (5.106) were co-transfected with 5 µg of either empty pcDNA3.1, pcDNA3.1-Myd88WT or pcDNA3.1-Myd88L252P vectors, plus 100 ng pRL-TK Renilla luciferase expression vector (Promega Corporation, Madison, WI) and 5 µg of either the 3X-κB-L vector with three copies of the major histocompatibility complex (MHC) class I κB element or its mutated counterpart, the 3X-mutκB-L vector (21 (link)) using Amaxa L013 program (AMAXA Biosystems, Cologne, Germany). Twenty four hours after transfection, cells were lysed and luciferase activities were measured using the Dual-Luciferase Reporter Assay System and the Turner Designs TD-20/20 Luminometer (Promega Corporation, Madison, WI).
3
Dual Luciferase Assay for Transcriptional Regulation
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293T cells grown in 48-well plates were transfected with pGL3-Basic vector or the reporter plasmids, or mutant reporter plasmids and pRL-TK Renilla luciferase expression vector (Promega) as an internal control as well as human DLX3 overexpressing plasmid (pcDNA3.1-Dlx3). After 48 h, the cells were collected and lysed. Luciferase activities were measured according to the Dual Luciferase reporter assay system (Promega). Relative luciferase activities were determined by normalizing each Firefly luciferase activities to Renilla luciferase activities. All experiments were conducted at least three times.
4
VEGF-C Promoter Activity Regulation
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NOZ cells were seeded at a density of 2×105 cells per well in 12-well plates. DNA transfection was performed in by use of Lipofectamine 2000 (Life Technologies; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocol. The small interfering (si)RNAs for NF-κB and AP-1 were 5'-CUCAAGAUCUGCCGAGUGA-3' and 5'-CCTCAGCAACTTCAACCC-3', respectively, and were synthesized by Beyotime Institute of Biotechnology; 100 pmol of each siRNA were transfected into the cells. At 48 h after transfection, the mRNA and proteins were extracted. NOZ cells were also co-transfected with the siRNAs and different VEGF-C promoter vectors, and cells were lysed 48 h after transfection. A total of 20 µg of cell lysate was used for the detection of intracellular luciferase activity, following the manufacturer's protocol of Dual-Luciferase® Reporter (DLR™) Assay system (Promega Corporation). The Renilla luciferase expression vector pRL-TK (Promega Corporation) was used for normalization and the promoter-less vector pGL3-Basic served as the negative control. Luminescence measurement was performed on a luminometer (Orion II Microplate Luminometer; Berthold Detection Systems GmbH). Each transfection was performed in duplicate and data were expressed as the mean ± standard deviation of three separate experiments.
5
Validating miR-140-5p Binding to SNHG16
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The fragment from SNHG16 containing the putative binding sites for miR-140-5p was amplified by PCR and cloned into the psiCHECK-2 vector (Promega, Madison, WI, USA), which was named SNHG16-WT. Point mutations in the binding seed regions were created by Quick Change Site-Directed Mutagenesis Kit (Agilent, Roseville City, CA, USA), and the resultant product was named SNHG16-MUT. A total of 3×104 HepG2 cells were seeded on 12-well plates and then cotransfected with SNHG16-WT or SNHG16-MUT, together with the Renilla luciferase expression vector pRL-TK (Promega) and miR-140-5p mimics or mimics control using Lipofectamine 2000. The activities of firefly luciferase and Renilla luciferase were measured 48 hours after transfection using the dual luciferase reporter assay system (Promega). Renilla luciferase was detected for data normalization.
6
Analysing NFκB-dependent Transcription
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Promoters containing four sequential copies of defined κB sites (5′-GGGAATTTCC-3′, 5′-GGGACTTTCC-3′, 5′-GGGGATTTCC-3′, 5′-GGGGCTTTCC-3′) were cloned into the pTAL-Luc vector (Clontech). WT and NFKB1S80A HEK293T cells were co-transfected with 100 ng pTAL-(4xκB) Luc vector and 10 ng Renilla luciferase expression vector pRL-TK (Promega). Twenty four hours post transfection, cells were cultured with or without 10 ng/ml TNFα for 8 h before harvest. Luciferase activities of whole cell lysates were analysed using the Dual-Luciferase Reporter Assay System (Promega). The ratio of firefly to Renilla luciferase activity was used to normalise for transfection efficiency across all samples.
7
Transfection Assay of Recombinant Plasmids
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Embryonic kidney (HEK-293), glioma (U87), and human neuroblastoma (SH-SY5Y) cell lines were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai) and cultured in a humidi ed 37 ℃ environment at 5% CO 2 . HEK-293 cells and U87 cells were cultured in HyClone® DMEM high glucose medium containing 10% fetal bovine serum (Thermo Fisher Scienti c, Massachusetts, USA), while SH-SY5Y cells were cultured in HyClone® DMEM/F-12 mixed medium containing 15% fetal bovine serum. When the cell density reaches 90% or higher, the transfection experiment was carried out. The cells were inoculated on 24-well plates with 2 × 10 5 cells per well. According to the manufacturer's agreement (Invitrogen, California, USA), the pGL3 recombinant plasmid containing 4 haplotypes and 8 truncated fragments was co-transfected with Lipofectamine® 3000 reagent and Renilla luciferase expression vector pRL-TK (Promega). After 24 hours of culture, the cells were collected. Finally, re y luciferase activity (LUC) and renal cell luciferase activity (TK) were measured. In each experiment, recombinant vectors were represented in triplicate and the experiment was repeated three times.
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