The promoter regions of mouse Postn gene were amplified from the mouse genomic DNA template and inserted into pGL4.15 empty vectors (Promega). Mutant promoters were generated using a PCR mutagenesis kit (Toyobo). All of the transient transfections were conducted using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For luciferase reporter assays, cells were seeded in 24-well plates and transfected with the indicated plasmids. Renilla luciferase pRL-SV40 (Promega) was used to normalize the luciferase activity, which was further measured using the dual-luciferase reporter assay system (Promega).
Prl sv40 renilla luciferase
Manufactured by Promega
Sourced in United States
The PRL-SV40 Renilla luciferase is a lab equipment product that serves as a reporter gene for monitoring gene expression. It utilizes the Renilla luciferase enzyme, which catalyzes the oxidation of coelenterazine to produce bioluminescence.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Pcr mutagenesis kit, by Toyobo (1 mentions)
Dual glo luciferase assay, by Promega (1 mentions)
Pgc1α, by Addgene (1 mentions)
Rspo3, by Genentech (1 mentions)
Chir99021, by STEMCELL (1 mentions)
X tremegene 9, by Roche (1 mentions)
Renilla luciferase, by Promega (1 mentions)
Hygromycin, by Corning (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
11 protocols using prl sv40 renilla luciferase
1
Cloning and Mutating Postn Promoter
2
NFAT-Luciferase Reporter Assay
3
Luciferase Assay for hOGG1 Promoter
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The luciferase reporter assay was used to detect the effect of SNP rs125701 on hOGG1 promoter activity. MGC-803 and BGC-823 cells were cultured in 24-well plates and transfected with 800 ng of recombinant plasmids using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). The 10 ng Renilla luciferase pRL-SV40 (Promega, Madison, WI, USA) was simultaneously cotransfected into cells per well as internal control. After 24 hours transfection, the cells were lysed and measured by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Relative luciferase activity was estimated by the ratio of Firefly and Renilla fluorescent intensity. Each transfection was carried out in independent triplicate.
4
STAT3 Promoter Regulation by ZNF831
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Cells were seeded in 24-well plates and human STAT3 promoter (−2000 to +200) luciferase reporter plasmid was transiently transfected with ZNF831 expression plasmids into cells. Renilla luciferase pRL-SV40 (Promega) was used to normalize the luciferase activity, which was further measured using the dual-luciferase reporter assay system (Promega).
5
Luciferase Reporter Assays for PGC1α
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Gal4-PGC1α, UAS-TATA luciferase, G6Pase luciferase, PEPCK luciferase, PGC1α-10Kb promoter luciferase, Cyt-C luciferase, NRF1 × 4 luciferase, PGC1α, cyclin D1, and cyclin D1K112E have been described previously and were obtained from Addgene (Cambridge, MA) (33 (link),34 (link),36 (link),44 (link)). pRL-SV40 renilla luciferase was used as a transfection control (Promega). Cos7 cells were transfected with DNA as indicated in the figure legends by using SuperFect or Attractene for 3 h. Cell extracts were prepared and luciferase activity was measured as previously described (47 (link)). Mutation of T298 and S312 of PGC1α was accomplished using site-directed mutagenesis. The resulting product was sequenced for fidelity and subcloned into pcDNA3.1.
6
Wnt Pathway Activation Assay
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HEK293 cells transfected stably with a TOPbrite firefly luciferase Wnt reporter and pRL-SV40 Renilla luciferase (Promega, E2231) were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum. For luciferase assay, cells were stimulated with CHIR99021 (StemCell Technologies, 72052), recombinant mouse WNT3A protein (R&D Systems, 1324-WN-002/CF) and the EC50 of RSPO3 (10.5 pM, Genentech) or the various PROTABs at either 1 or 10 μg ml−1. Following 16 h treatment, luciferase activity was detected using the Promega Dual-Glo system (Promega, E2920) according to the manufacturer’s instructions. Data were analysed as the ratio of firefly/Renilla luciferase activity.
7
Wnt Signaling Assay in HEK293 Cells
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Wnt signaling assays were performed in HEK293 cells with stably integrated firefly-luciferase-based Wnt reporter (TB) (77 (link)) and pRL-SV40 Renilla luciferase (Promega) as described in the SI Appendix .
8
Luciferase Assay for Interferon Response
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For luciferase reporter gene-transactivation assays, cells were co-transfected with the plasmid pGAS-Luciferase or pISRE-Luciferase (0.5 μg; Stratagene) and pRL-SV40-Renilla luciferase plasmid (0.03 μg, Promega) using X-tremeGene 9 (Roche Applied Science). After 24 h, transfected cells were treated for 12 h with IFN-γ (100 U/ml) or IFN-β (1,000 U/ml) and then split into aliquots for analysis of luciferase activity. The level of renilla luciferase activity was used to correct for transfection efficiency.
9
BMP Signaling Activation by GDF6
10
Wnt Reporter Assay in HEK293 Cells
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HEK293 cells with stably integrated firefly-luciferase-based Wnt reporter (TOPbrite) (66 (link)) and pRL-SV40 Renilla luciferase (Promega) were maintained in a 5% CO2 humidified incubator at 37 °C in DMEM with nutrient mixture F12 (50:50), 10% (v/v) FBS, 2 mM GlutaMAX (Gibco), and 40 μg/mL hygromycin (Cellgro). Cells were grown for at least 24 h before any experiments. 20,000 to 40,000 cells/well in 50 μL medium were seeded in each well of clear-bottom white polystyrene 96-well plates (Falcon) and incubated for 24 h. The cells were then transfected with 0 to 25 ng of Wnt or Wnt chimera-expressing constructs (Wnt3a_1, Wnt5a_1, and Wnt3a_haXWnt8) mixed with FuGENE HD in 10 μL OptiMEM, followed by incubation for 24 to 48 h. Treatment with peptides or LRP6 Fabs was done for 6 h before the assay measurement. Readout was obtained with 50 μL of Dual-Glo Luciferase Assay system (Promega) according to the manufacturer’s instructions on a Perkin Elmer EnVision multilabel reader. The ratios of firefly luminescence to Renilla luminescence were calculated, and in some instances normalized to control (nontreated) samples. Cell lines were tested for mycoplasma contamination and authenticated by single-nucleotide polymorphism analysis.
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