For the ENC1-P luciferase reporter gene, the 1851-bp region of the ENC1 promoter was subcloned into the XhoI and HindIII sites of the pGL3-Basica vector (Promega Corporation, Madison, WI, USA). To generate the ENC1 promoter enhancer (PE) luciferase reporter genes, the 1899-bp region of the ENC1-E1 enhancer, the 1838-bp region of the ENC1-E2 enhancer, the 1533-bp region of the ENC1-E3 enhancer, and the 1999-bp region of the ENC1-E4 enhancer were subcloned into ENC1. The luciferase reporter genes ENC1-E1, ENC1-E2, ENC1-E3, and ENC1-E4 plasmids were generated using 4-Promoter luciferase reporter gene with the SalI/BamHI restriction enzyme site. To confirm the TCF4 binding sites on the ENC1-E2 enhancer, a 241 bp region containing the wild-type (WT) TCF4 motif-binding sequence (from chr5: 74687892-74684243) was inserted into the SalI and BamHI sites of the pGL4 vector (downstream of the luciferase) and the minimal promoter (Promega) to produce the TCF4 luciferase reporter gene (pGL4-WT). To generate the pGL4-mutant (Mut) plasmid, mutant TCF4 binding sites from GTGGTGGTTT to GTGAACGTT were constructed by PCR-based site-directed mutagenesis.
Minimal promoter
Manufactured by Promega
Sourced in United States
The Minimal promoter is a short DNA sequence that acts as a core promoter, providing the necessary elements for transcription initiation. It serves as a fundamental component in gene expression studies and recombinant DNA technologies.
Automatically generated - may contain errors
Lab products found in correlation
Pgl3 basica vector, by Promega (1 mentions)
Lipofectamine plus reagent, by Thermo Fisher Scientific (1 mentions)
Prl cmv renilla luciferase, by Promega (1 mentions)
Td 20 20 luminometer, by Promega (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Tnf α, by R&D Systems (1 mentions)
2 protocols using minimal promoter
1
Luciferase Reporter Assay for ENC1 Promoter and Enhancers
2
Dual Luciferase Reporters for GR and NF-κB
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The Firefly Luciferase glucocorticoid-responsive reporters TAT-Luciferase (Luc) and MMTV-Luc, and NF-κB reporter × 3 κB-Luc were described previously.28 (link) The transfection efficacy was normalized using co-transfections with pRL-CMV-Renilla luciferase (RL) under minimal promoter (Promega, Madison, WI, USA). 3PC cells were transfected in 24-well plates (at least three wells/experimental group) using Plus Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Each well contained 0.33 μg of the plasmid DNA. Twenty four hours after transfection, cells were treated with vehicle (0.01% acetone), CpdA (10−6−10–5 M) or FA (10−6 M) for 24 hours. To activate NF-κB, we used TNF-α (10 ng/mL; R D Systems, Minneapolis, MN, USA). Cells were treated with TNF-α and GR ligands simultaneously for 24 hours. The Firefly and RL activity was measured using TD20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA) following the protocol for Dual Luciferase reporter assay (Promega).
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