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Prl tk vector expressing renilla luciferase

Manufactured by Promega
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The PRL-TK vector is a plasmid construct that expresses the Renilla luciferase reporter gene. The Renilla luciferase gene is driven by the PRL (Rous Sarcoma Virus Long Terminal Repeat) promoter and the thymidine kinase (TK) polyadenylation signal. This vector can be used for various applications that require the expression of the Renilla luciferase reporter.

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6 protocols using prl tk vector expressing renilla luciferase

1

Measuring BCL-2 Promoter Activity in Glioblastoma Cells

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The BCL-2 reporter plasmid (Addgene #15381) containing the BCL-2 promoter region from ATG to −3934 was a kind gift from Linda Boxer. A luciferase reporter assay system (Promega, Madison, WI, USA) was used to determine the luciferase activity of the BCL-2 promoter. U87/TMZ and U251/TMZ cells were seeded in a 96-well plate (8 × 103 cells/well) and transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, Burlington, MA, USA) and 20 ng pRL-TK vector expressing Renilla luciferase (Promega), following the recommended protocol using Lipofectamine 3000 (Thermo Scientific). Cells were harvested 48 h later for analysis using a dual-luciferase reporter assay system (Promega). Luciferase activity was measured using a PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer). Luciferase intensity was normalized to Renilla luciferase activity to adjust for transfection efficiency.
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2

Wnt/β-catenin Pathway Activation by G. lucidum

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MG63 and U2-OS cells were, respectively, inoculated in a 96-well plate (5 ×
103 cells/well) and incubated for 24 hours, then transfected with
200 ng TOP-Flash/FOP-Flash (Millipore, Burlington, MA), and 20 ng pRL-TK vector
expressing Renilla luciferase (Promega, Madison, WI), following the recommended
protocol using Lipofectamine 3000 (Thermo Scientific), for another 24 hours.
Subsequently, cells were divided into 2 groups: G lucidum (0,
100, or 200 µg/mL for 24 hours) or CHIR-99021 (inhibitor of GSK-3α/β, Selleck,
Houston, TX) + G lucidum (cells were pretreated with 10 µm
CHIR-99021 for 24 hours to activate the Wnt/β-catenin signaling pathway), and OD
values of the TOP flash and FOP flash were detected by a dual-luciferase
reporter assay system (Promega) from cell lysates. Activity of the Wnt/β-catenin
signaling pathway was reflected by the TOP/FOP ratio.
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3

Validating miR-539 Target Gene DLX1

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The target gene of miR‐539 was conducted using the microRNA.org. Dual luciferase reporter gene assay was conducted to confirm whether DLX1 was the target gene of miR‐539. The 3′‐untranslated region (3′UTR) of the DLX1 gene was cloned into pmirGLO vector (E1330, Promega Corp., Madison, WI, USA) and was given the name pDLX1 wild‐type (Wt). Site‐directed mutagenesis was performed on the potential binding sites of miR‐539 and the pDLX1‐mutant (Mut) vector was also constructed. pRL‐TK vector expressing Renilla luciferase (E2241, Promega Corp., Madison, WI, USA) was constructed to serve as an internal control. The miR‐539 mimic or the NC plasmids were transfected into cells together with pDLX1‐Wt or pDLX1‐Mut. After 48 hours of transfection, the cells were collected and luciferase activity was detected according to the instructions of the luciferase assay kit (GeneCopoeia, Inc, Rockville, Maryland, USA).
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4

Modulating Wnt/β-catenin Signaling

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U2-OS Cells were inoculated in a 96-well (5×103 cells/well) and were incubated for 24 h, and then transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, MA, USA), and 20 ng pRL-TK vector expressing Renilla luciferase (Promega, WI USA) following the recommended protocol using Lipofectamine 3000 (Thermo Scientific, MA, USA) for another 24 h. Subsequently, the assays were divided into four groups: resveratrol (0, 6 or 12 μg/ml resveratrol for 24 h), CHIR-99021 (inhibitor of GSK-3α/β, Selleck, TX, USA) + resveratrol (the cells were pretreated with 10 μm CHIR-99021 for 24 h to activate the Wnt/β-catenin signaling pathway); lentivirus infection (shCx43, NTC and Blank), and lentivirus infection + XAV939 [(inhibitor of β-catenin, Selleck, TX, USA), the cells were treated with 10 μm XAV939 for 24 h to suppress the Wnt/β-catenin signaling pathway]. The OD values of the TOP flash and the FOP flash were detected by a dual-luciferase reporter assay system (Promega, WI, USA) from cell lysates, and the TOP/FOP ratio reflected the activity of the Wnt/β-catenin signaling pathway.
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5

Bdnf Promoter-Driven Luciferase Assay

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Cell transfections were performed on HEK293 cells that were maintained in DMEM supplemented with 10% fetal bovine serum. The cells were co-transfected with 2 µg Bdnf promoter promoter sequences to drive luciferase expression and 2 µg pRL-TK vector expressing Renilla luciferase (Promega) using Fugene 6 (Roche) according to the manufacturer’s instructions. Forty-eight h after transfection, cells were incubated in the presence of experimental treatments for 6 h. Luciferase activity was quantified using a Dual-Luciferase-Reporter System (Promega). Luciferase activity in each sample was normalized to the internal control renilla luciferase activity. Luminescence was expressed in an arbitrary scale as relative light units (RLU).
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6

HCMV UL54 Promoter Analysis

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The pUL54-luciferase indicator plasmids pUL54-0.4, bearing the entire HCMV UL54 promoter, and pUL54-0.15, containing UL54 promoter sequence from -150 to +15 relative to the transcription start site, were previously reported (Gariano et al., 2012) . The pSGIE72 (IE1 expression construct), the luciferase indicator construct phTS-243/+30 [that contains a portion of the promoter of the human thymidylate synthase gene (-243 and +30 relative to the AUG start codon)] were previously described (Gribaudo et al., 2002) . pSG5 vector was purchased from Stratagene/Agilent Technologies and the pRL-TK vector expressing Renilla luciferase was from Promega.
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