SiHa cells were transfected with 100 ng p65-luciferase reporter construct (containing five copies of the p65-binding site, Promega) or 200 ng p53-luciferase reporter construct (containing thirteen wildtype p53 binding sites, Addgene plasmid #16442, Addgene Plasmid Repository [33 (link)] and 10 ng pRL-TK (encoding Renilla luciferase, Promega), using Genecellin transfection reagent (Celtic Molecular Diagnostics). The following day cells were treated with 5 μM INI-43 for 2 h, followed by 30 μM cisplatin for 24 h, and luciferase activity assayed using the Dual-Luciferase Report assay system (Promega), according to the manufacturer’s instructions. Luciferase readings were measured using the Veritas™ microplate luminometer (Promega) and normalised to Renilla luciferase from the same extract.
P65 luciferase reporter construct
Manufactured by Promega
The P65-luciferase reporter construct is a plasmid that contains the firefly luciferase gene under the control of the NF-κB (p65) response element. This construct can be used to monitor NF-κB transcriptional activity in cells.
Automatically generated - may contain errors
2 protocols using p65 luciferase reporter construct
1
Measuring p65 and p53 Transcriptional Activity
2
Transcriptional activity assay for p65 and p53
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SiHa cells were transfected with 100 ng p65-luciferase reporter construct (containing ve copies of the p65-binding site, Promega) or 200 ng p53-luciferase reporter construct (containing thirteen wildtype p53 binding sites, Addgene plasmid #16442, Addgene Plasmid Repository(26) and 10 ng pRL-TK (encoding Renilla luciferase, Promega), using Genecellin transfection reagent (Celtic Molecular Diagnostics). The following day cells were treated with 5 µM INI-43 for 2 hours, followed by 30 µM cisplatin for 24 hours, and luciferase activity assayed using the Dual-Luciferase Report assay system (Promega), according to the manufacturer's instructions. Luciferase readings were measured using the Veritas™ microplate luminometer (Promega) and normalised to Renilla luciferase from the same extract.
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