Subconfluent cells cultures were transfected in triplicate using the jetPEI transfection reagent (PolyPlus Transfection, Illkirch, France), following the manufacturer's guidelines. The reporter plasmids pTOPFLASH, pFOPFLASH, 4xwtCBF1Luc, 4xmtCBF1Luc, NF3, and that harbouring the −2235/+112 region of the DKK1 promoter upstream of the Firefly luciferase gene have been previously described.19 , 20 , 21 , 22 The reporter plasmids harbouring wild‐type and mutant PKP2 promoter and enhancer sequences are described above. The expression plasmid pFLAG‐CMV.5.1‐Plakophilin2a‐flag encoding Plakophilin‐2 was purchased from Addgene (Cambridge, MA). The expression plasmids pMT23‐β‐catenin, pCDNA3‐TCF4‐VP16 and pCDNA3‐p65 have been described elsewhere.22 , 23 , 24 The expression plasmid pCDNA3‐ICND was a generous gift from Keith R. Brennan (University of Manchester, UK). A Renilla luciferase plasmid (pRL‐TK) was used in all experiments as an internal control. Firefly and Renilla luciferase activities were measured separately using the Dual Luciferase reagent kit (Promega) and a GloMax 96 microplate luminometer (Promega).
Dual luciferase reagent kit
Manufactured by Promega
The Dual Luciferase Reagent Kit is a laboratory assay used to measure the activity of two different luciferase reporter genes simultaneously within a single sample. The kit provides reagents to lyse cells and detect the activities of both firefly and Renilla luciferase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Ptk ren, by Promega (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Jetpei transfection reagent, by Polyplus Transfection (1 mentions)
3 protocols using dual luciferase reagent kit
1
Wnt Signaling Pathway Reporter Assays
2
TBEV replicon RNA transfection assay
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Cells were seeded at 1.5 × 105 cells per well in 96-well plates (n = 3) and transfected with in vitro transcribed TBEV replicon RNA (100 ng/well) and 10 ng of pTK-Ren (Promega) using Lipofectamine 2000 (Thermo Fisher). Cells were harvested in passive lysis buffer (Promega) at 12 to 72 hpt, and luciferase activity was measured using the Dual Luciferase reagent kit and GloMax multidetection system (Promega). Firefly luciferase readings from the TBEV replicons were normalized to the renilla values (pTK-Ren) of each sample.
3
TBEV Replicon Assay in PS Cells
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PS cells were seeded at 1.5 x105 cells per well in 96-well plates (n = 3) and transfected with in vitro transcribed TBEV replicon RNA (100 ng/well) and 10 ng of XbaI linearized pTK-Ren (Promega) using Lipofectamine-2000 (Thermo Fisher Scientific). Cells were harvested in passive lysis buffer (Promega) at 12 to 72 hpt. Luciferase activity was measured using the Dual Luciferase reagent kit and GloMax multi detection system (Promega). pTK-Ren (Promega) expressing Renilla luciferase (RLuc), was included as an expression control. Quantitative data were obtained from three independent experiments.
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