For the NF-κB transcriptional activity assays, HUVECs were transfected with a cis-reporter plasmid containing 5 copies of consensus NF-κB sequences linked to a minimal E1B promoter-luciferase gene (pNF-κB Luc; Stratagene, La Jolla, California) using Lipofectamine 2000, following the manufacturer's instructions. The pRL-SV40 Renilla luciferase control reporter vector (Promega Corporation, Madison, Wisconsin) was used as an internal control. Twenty-four hours after transfection, the cells were exposed to TP at the indicated concentrations for 1 hour, followed by incubation with ox-LDL (50 µg/mL) for an additional 1 hour. Afterward, the cells were harvested and lysed with a cell lysis buffer (Cell Signaling Technology Inc, Danvers, Massachusetts). Firefly luciferase activity was assayed using a dual-luciferase reporter assay system (Promega Corporation) with a TD-20/20 luminometer (Turner BioSystems). To express relative luciferase activity, Renilla luciferase activity was used to normalize firefly luciferase activity.
Prl sv40 renilla luciferase control reporter vector
Manufactured by Promega
Sourced in United States
The PRL-SV40 Renilla luciferase control reporter vector is a plasmid that expresses the Renilla luciferase gene under the control of the SV40 early promoter. It can be used as a control reporter vector in gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Td 20 20 luminometer, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pgl3 promoter vector, by Promega (1 mentions)
Victor 3, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Envision multilabel plate reader, by PerkinElmer (1 mentions)
Pfn11a bind flexi vector, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay kit, by Promega (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
6 protocols using prl sv40 renilla luciferase control reporter vector
1
NF-κB Transcriptional Activity Assay in HUVECs
2
Luciferase Assay for GRN 3' UTR
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The wild‐type and mutant 3′ UTR sequences of Rno GRN were synthesized by GenePharma (Shanghai, China) and subcloned into the pGL3 promoter vector containing the luciferase reporter (Promega, Madison, WI, USA). The recombinant plasmids were respectively named as, pGL3‐GRN 3′ UTR‐WT, pGL3‐GRN 3′ UTR‐MU1 and pGL3‐GRN 3′ UTR‐MU2. SW1353 or HEK293T cells were seeded in 24‐well plates. When the cells reached about 70% confluences, each recombinant plasmid (200 ng) and miR‐29b‐3p mimic (200 ng) were cotransfected into the cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's direction. The pGL3 promoter vector (200 ng) was used as the control. To normalize the activity of fly luciferase, we cotransfected the pRL‐SV40 Renilla luciferase control reporter vector (Promega) into SW1353 or HEK293T cells. Experiments were performed in quadruplicate and repeated at least three times.
3
Noxa Promoter Region Analysis
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pGL10.4 (Promega, USA) was used to study the impact of PRIMA-1MET on the Noxa promoter region. Three plasmids containing NOXA promoter region from + 198 to − 157 were designed. In two of the plasmids, additional mutations were inserted in p53 binding site and CREB binding site. All plasmids were prepared by GenScript (USA). The pRL-SV40 Renilla luciferase control reporter vector (Promega, USA) was used as a normalization control at a 1:10 weight ratio. Plasmids were transfected into SK-N-SH cells using X-tremeGENE HP DNA Transfection Reagent (Roche, Switzerland) according to manufacturer’s recommendations. The Dual-Luciferase Reporter Assay System (Promega, USA) and Victor3 (Thermo Fisher Scientific, USA) were used to measure luciferase activity (5 s shaking at medium speed prior to 1 s measurement time without the use of filters).
4
Measuring NF-κB Transcriptional Activity
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To determine NF-κB transcriptional activity, HUVECs were transfected with a cis-reporter plasmid containing 5 copies of consensus NF-κB sequences linked to a minimal E1B promoter-luciferase gene (pNF-κB Luc; Stratagene, LaJolla, CA, USA) using LipofectAMINE 2000, according to the manufacturer’s protocol. pRL-SV40 Renilla luciferase control reporter vector (Promega Corporation, Madison, WI, USA) was co-transfected as an internal control. A total of 24 h after transfection, the cells were treated with LPS in the presence or absence of triptolide at the indicated doses. After 1 h, the cells were harvested and lysed with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Firefly luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) with a TD-20/20 luminometer (Turner BioSystems, Sunnyvale, CA, USA). Relative luciferase activity is presented as a ratio of firefly luciferase activity to Renilla luciferase activity.
5
Measuring RORγt Transcriptional Activity
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To generate the RORγt-GAL4 fusion protein, human RORγt (from 97 to 516) was PCR-amplified from pCDNA2-FLAG-RORγt. The PCR product was ligated into the pFN11A (BIND) Flexi vector (Promega, Madison, WI) containing the yeast GAL4 DNA-binding domain upstream of the cloning site. This vector also expresses the Renilla luciferase under the control of SV40 promotor, allowing normalization for differences in transfection efficiency. The recombination plasmid was named RORγt-GAL4. The vector, pGL4.31 [luc2P/GAL4UAS/Hygro] (Promega, Madison, WI), contains five tandem GAL4 binding sites upstream of a minimal TATA box, which is upstream of a firefly luciferase gene that acts as a reporter for interactions between protein and the ligand.42 (link) The pRL-SV40 Renilla luciferase control reporter vector was purchased from Promega (Madison, WI).
For luciferase activity assay, HEK293T cells were co-transfected with 2 μg ROR-GAL4, 2 μg pGL4.31 and 100 ng pRL-SV40 in a 6 cm dish using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, the cells were incubated with a variety of concentrations of compounds. After 24 h treatment, the cells were lysed with passive lysis buffer and luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI) using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA).
For luciferase activity assay, HEK293T cells were co-transfected with 2 μg ROR-GAL4, 2 μg pGL4.31 and 100 ng pRL-SV40 in a 6 cm dish using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, the cells were incubated with a variety of concentrations of compounds. After 24 h treatment, the cells were lysed with passive lysis buffer and luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI) using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA).
6
Quantifying STAT6 Transcriptional Activity
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The p4xSTAT6-Luc2P luciferase reporter plasmid encoding 4xSTAT6 binding sites [TTCCCAAGAA] was obtained from Addgene (catalogue no. 35554) and used to assess STAT6 promoter activity. The pRL-SV40 Renilla luciferase control reporter vector was obtained from Promega (catalogue no. E2261) and was used in cotransfection studies with p4xSTAT6-Luc2P for internal normalization. HEK293 cells were seeded in 96-well plates overnight at a density of 30,000 cells per well and transfected with 20ng of plasmids encoding either GFPtagged WT or p.E372K STAT6. The cells were further transfected with 100ng of p4xSTAT6-Luc2P and 4ng the pRL-SV40 vectors using Fugene HD transfection reagent (Promega, catalogue no. E2311), according to manufacturer's recommendations. The next day, transfected cells were stimulated with 100ng/mL of IL-4 (PeproTech, catalogue no. 200-04), and transfected but otherwise untreated cells were used as a control group. Firefly and Renilla luciferase activities, as indicated by relative luminescence units (RLU), were determined using Dual-Glo luciferase assay kits (Promega, catalogue no. E2920), according to the manufacturer's instructions. Luciferase activity was measured on BioTek Synergy HTX Multi-Mode Microplate Reader.
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