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Pg5luc reporter

Manufactured by Promega
Sourced in United States

The PG5Luc reporter is a plasmid-based system that contains the firefly luciferase gene under the control of a synthetic promoter. It is designed for the measurement of transcriptional activity in various cell lines.

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5 protocols using pg5luc reporter

1

Transient Transfection of Mutant PPARγ Plasmids

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293T cells(ATCC, USA) were maintained in DMEM containing 10% fetal bovine serum (FBS) and were transiently transfected using Lipofectamine 2000 reagent (Invitrogen, USA). All mutant PPARγ plasmids were created using the Quick-Change site-directed mutagenesis kit (Stratagene, USA). Twenty-four-well plates were plated 24 hours prior to transfection (5 × 104 cells per well). For the Gal4-driven reporter assays, the cells were transfected with 200 ng of Gal4-LBDs of various nuclear receptors and 200 ng of pG5Luc reporter (Promega, USA). Ligands were added five hours after transfection. Cells were harvested 24 h later for the luciferase assays. Luciferase activities were analyzed as the the instruction of CheckMate™ Mammalian Two-Hybrid System (Promega, USA).
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2

Optogenetic Regulation of Luciferase Expression

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The day before transfection, cells were trypsinized and transferred into wells of polystyrene 96-well culturing plates (Costar). Ten µL of a water solution containing 30 ng of the mutated Gal4-FusionRed-p65 construct, 0.1 µg of the pG5-Luc reporter (Promega), encoding the secreted Gaussia luciferase, and 0.2 µg pGl3-Basic vector was mixed with 1.5 µL of 1 mg/mL solution of PEI, pH = 7.0. The mixture was incubated for 10 min at room temperature for the efficient formation of DNA-PEI complexes and added directly to cells in plate wells containing 100 µL of the cultured medium. After transfection, cells were illuminated for 24 h with either blue pulsed light (array of 448 nm light-emitting diodes (LEDs), 30 s ON, 5 min OFF) or yellow-orange pulsed light (567 nm LED array, 30 s ON, 5 min OFF) at a light power density of 100 mW/cm2.
For measurements of Gaussia luciferase activity, 1 µL of cell culture medium was mixed with 100 µL of 2 µM coelenterazine (Sigma, St. Louis, MO, USA) in wells of 96-well reflective bottom plates for bioluminescent assays (Stellar Scientific, Owings Mills, MD, USA). The quantification of bioluminescence was performed using a Victor X-5 plate reader (Perkin Elmer, Waltham, MA, USA). The contrast of 448 nm/567 nm reporter expression was calculated as luciferase activity in samples under illumination with 448 nm light divided by that at 567 nm light.
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3

Quantifying Nuclear Receptor Activity

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HEK293T cells were cotransfected with pG5-luc reporter (Promega) together with the plasmids encoding different NR-LBDs fused with the DNA-binding domain of Gal4. One day after transfection, cells were treated with DMSO, Z compounds or ligands specific for each nuclear receptor. After 12 hours, cells were lysed by passive lysis buffer. Firefly and Renilla luciferase activities were quantitated using the Dual-Luciferase Reporter Assay System (Promega, E1960). Transfection and expression efficiency was normalized to renilla luciferase activity.
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4

Transient Transfection and Luciferase Assay for Nuclear Receptor Activity

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The HEK–293T cells were maintained in DMEM containing 10% fetal bovine serum, and were transiently transfected using Lipofectamine 2000 (Invitrogen). All mutant PPARα and PPARδ plasmids were created using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The resulting plasmids were confirmed by DNA sequencing. Before 24 h of transfection, 24-well plates were plated (5 × 104 cells per well). For the nuclear receptor luciferase reporter assay, the cells were transfected with 200 ng Gal4-LBDs of various nuclear receptors and 200 ng of pG5Luc reporter (which contained five GAL4 binding sites) (Promega, Madison, WI, USA) [37 (link),38 (link)]. For native promoter-reporter assays, the cells were co-transfected with plasmids encoding full-length PPARs and a peroxisome proliferator hormone response element (PPRE). Ligands were added 5 h after transfection. Cells were harvested 24 h later for the luciferase assays with the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The luciferase activities were normalized to renilla activity, co-transfected as an internal control. The dose curves were fitted by GraphPad Prism 8.
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5

Transactivation Assay of RXRα

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293T cells were cotransfected with pG5-luc reporter (Promega) together with the RXRα-LBD fused with the DNA-binding domain of Gal4. After 24 hours, cells were treated with DMSO, Z compounds, or 9-cis- RA. After 12 hours, cells were lysed by passive lysis buffer. Firefly and Renilla luciferase activities were quantitated using the Dual-Luciferase Reporter Assay System (Promega, E1960). Transfection and expression efficiency was normalized to Renilla luciferase activity.
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