The promoter of large yellow croaker SHP was cloned into the reporter plasmid (pGL3-basic vector, Promega, USA) by a ClonExpress II One Step Cloning Kit (Vazyme, China) (17 (link)). The expression plasmid of large yellow croaker NRF2 was stored in our laboratory. HEK 293T cells were seeded into 24-well plate (5 × 105 cells) and co-transfected with reporter plasmids (200 ng/well), phRL-CMV plasmid (20ng/well, Promega, USA), and expression plasmids (200-600 ng/well). After 24 h transfection, cells were collected and the luciferase activity was measured using a TransDetect double-luciferase reporter assay kit (TransGen Biotech, China).
Phrl cmv plasmid
Manufactured by Promega
Sourced in United States
The PhRL-CMV plasmid is a laboratory tool used for gene expression studies. It contains the Renilla luciferase (RLuc) reporter gene under the control of the cytomegalovirus (CMV) promoter, allowing for the expression and measurement of Renilla luciferase activity in transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter system, by Promega (2 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Transdetect double luciferase reporter assay kit, by Transgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipod293 in vitro dna transfection reagent, by SignaGen (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Nf κb luc, by Promega (1 mentions)
4 protocols using phrl cmv plasmid
1
Promoter Analysis of Large Yellow Croaker SHP
2
Cloning and Characterization of OCN Promoter
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To generate the OCN promoter reporter, the promoter sequence (1252 bp) upstream of the transcriptional start site of human BGLAP (encoding OCN protein) was cloned into luciferase reporter plasmid and verified with direct sequence (pGL-OCN). The 293 T cells (2 × 105 cells/well in 24-well plates) were transiently transfected with pGL-OCN, TAZ, constitutively active TAZ (TAZS89A), or TEAD4 plasmids and phRL-CMV plasmid (Promega) using lipofectamine 3000. Cells were lysed 24 h after transfection and assayed for firefly and Renilla luciferase activity using the Dual-Luciferase reporter system (Promega). The data are expressed as the ratios of firefly to Renilla activity.
3
Regulation of SOX2 Promoter Activity
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The promoter sequence (2065 bp) upstream of the transcriptional start site of human SOX2 was subcloned into a luciferase reporter plasmid and verified with direct sequencing. Two putative binding sites between TEAD4 and SOX2 were individually mutated using QuikChange® Lightning Site-Directed Mutagenesis Kits (Stratagene) and verified by direct sequencing. The 293 T cells were transiently transfected with pGL-SOX2, TAZ or TEAD4 plasmids and phRL-CMV plasmid (Promega) using lipofectamine 2000 (Invitrogen). Cells were harvested 24 h after transfection and assayed for Firefly and Renilla luciferase activity using the Dual-Luciferase reporter system (Promega). Data were presented as the ratios of Firefly to Renilla luciferase activity.
4
Assessing NF-κB Transcriptional Activity
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To assess the NF-κB transcriptional activity, we used the NF-κB–luciferase reporter construct (NF-κB-luc; Promega). HEK293T cells were transiently cotransfected with NF-κB-luc reporter plasmid and phRL-CMV plasmid (Promega) with or without the indicated constructs using the LipoD293 DNA In Vitro Transfection Reagent (SignaGen Laboratories). Cells were lysed and assayed for firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay System (Promega). The data were expressed as the ratio of firefly to Renilla activity.
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