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Phrl tk renilla luciferase reporter plasmid

Manufactured by Promega
Sourced in United States

The PhRL-TK Renilla luciferase reporter plasmid is a laboratory tool that contains the Renilla luciferase gene under the control of a thymidine kinase (TK) promoter. Renilla luciferase is an enzyme that catalyzes a bioluminescent reaction, producing light that can be measured and quantified. This plasmid is commonly used as a reporter system to study gene expression and regulatory mechanisms in various cell types and experimental settings.

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4 protocols using phrl tk renilla luciferase reporter plasmid

1

Dual-Luciferase Reporter Assay for SGTA and REIC/Dkk-3 Interaction

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For the MTH assay, the full-length open reading frame (ORF) of canine SGTA and REIC/Dkk-3 cDNA was cloned between the EcoRI and MluI sites of a pM GAL4 DNA-binding domain cloning plasmid (GAL4-DBD) and pVP16 transactivation domain cloning plasmid (VP16-AD) (Clontech Laboratories, Palo Alto, CA, USA), respectively. In addition, the full-length ORF of canine REIC/Dkk-3 cDNA was cloned between the XhoI and EcoRI sites of the pMACS KK HA(C) vector cloning plasmid (Miltenyi Biotec, Bergisch Gladbach, Germany). Primers were used in this study was described in Additional file 1: Table S1. Approximately 2 × 105 293 T cells per well in 24-well plates were co-transfected with 100 ng of pM, pVP16, pMACS KK HA(C), and pFR-Luc firefly luciferase reporter plasmid (Promega, Madison, WI, USA), and 0.2 ng of phRL-TK Renilla luciferase reporter plasmid (Promega). The cells were harvested 48 h after transfection, and the luciferase activity was measured using the dual-luciferase reporter assay system (Promega). The luciferase activity was normalized to the value of the Renilla luciferase activity [21 (link)].
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2

NFAT Transcription Factor Activation Assay

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A NFAT reporter assay was performed as described previously56 (link). In brief, cells were transfected with 10 μg pNFAT-luc (a gift from Dr. Gerald R. Crabtree, Stanford University) and 1 μg phRL-TK Renilla-luciferase reporter plasmid (Promega, Madison, WI) by electroporation. The cells were then sensitized with or without the anti-DNP IgE mAb (200 ng/ml) for 24 h. After removal of unbound IgE, the cells were stimulated with or without the anti-myc mAb (10 μg/ml) or DNP-BSA (30 ng/ml) for 6 h at 37 °C. The reporter activity was assessed by the Dual-Glo luciferase assay system (E2920, Promega) according to the manufacturer’s instructions.
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3

Mammalian Cell Two-Hybrid Assay for REIC/DKK-3 and SGTA Interactions

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For the mammalian cell two-hybrid assay, various lengths of human REIC/DKK-3 and SGTA cDNA were cloned into the BamHI and MluI sites of the pM GAL4 DNA-binding domain cloning plasmid (GAL4 DBD) and pVP16 transactivation domain cloning plasmid (VP16 AD) (Clontech Laboratories), respectively. The templates for each length of human REIC/DKK-3 and SGTA were generated by PCR amplification using appropriate primer pairs. Approximately 2 × 105 293T cells on 24-well plates were co-transfected with 100 ng of pM, 100 ng of pVP16, 100 ng of pFR-Luc firefly luciferase reporter plasmid (Promega) and 2 ng of phRL-TK Renilla luciferase reporter plasmid (Promega). The cells were harvested at 48 hours after transfection, and luciferase activity was measured using a dual-luciferase reporter assay system. Luciferase activity was normalized to Renilla luciferase activity [35 (link)]. DBD- and VP16-fused human SGTA constructs were introduced into 293T cells transfected with the REIC/DKK-3 constructs in the pMACS Kk.HA-C vector to examine the interference of SGTA-SGTA dimerization by REIC/DKK-3.
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4

Mammalian Cell Two-Hybrid Assay

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For the mammalian cell two-hybrid assay (MTH), WT and Y208C cIDH1 cDNA were cloned into the EcoRI and MluI sites of the pM GAL4 DNA-binding domain of the GAL4-DBD plasmid (pM) (Clontech Laboratories) and the pVP16 transactivation domain of the VP16-AD plasmid (pVP16) (Clontech Laboratories), respectively. Approximately 2×105 HeLa cells were placed in a 24-well plate and were co-transfected with 100 ng of pM, 100 ng of pVP16, 100 ng of pFR-Luc firefly luciferase reporter plasmid (Promega), and 2 ng of phRL-TK Renilla luciferase reporter plasmid (Promega). The cells were harvested 48 h after transfection, and luciferase activity was measured using a Dual-Glo Luciferase Assay System (Promega). Luciferase activity was normalized to that of Renilla luciferase activity.
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