The wild-type (TOP) and mutant (FOP) LEF/TCF reporters were cloned into pGL3 luciferase constructs (Promega). Twenty thousand cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of TOP or FOP flash, plus 1 ng of pRLTK Renilla plasmid (Promega), was transfected into cells using the Lipofectamine 3000 reagent according to the manufacturer’s recommendation. Luciferase and Renilla signals were measured 24 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. The results were calculated as the ratio of specific TOP-Flash to nonspecific FOP-Flash relative Renilla luciferase units (RLU).
Pgl3 luciferase construct
Manufactured by Promega
Sourced in United States
The PGL3 luciferase constructs are a set of reporter gene plasmids designed for various cell-based assays. These constructs contain the firefly luciferase gene under the control of different promoters, allowing for the measurement of transcriptional activity in cells. The core function of these constructs is to provide a reliable and quantitative means of monitoring gene expression in a variety of experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Prl tk renilla plasmid, by Promega (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Dual luciferase reporter gene kit, by Promega (1 mentions)
Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions)
Dual luciferase system, by Promega (1 mentions)
Prl tk vector, by Promega (1 mentions)
6 protocols using pgl3 luciferase construct
1
Analyzing Wnt Signaling Activity
2
Luciferase Assay for Wnt Signaling
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The wild-type (TOP) and mutant (FOP) LEF/TCF reporters were cloned into pGL3 luciferase constructs (Promega). Twenty thousand cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of TOP or FOP flash, plus 1 ng of pRL-TK Renilla plasmid (Promega), was transfected into cells using the Lipofectamine 3000 reagent according to the manufacturer's recommendation. Luciferase and Renilla signals were measured 24 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. The results were calculated as the ratio of specific TOP-Flash over non-specific FOP-Flash relative renilla luciferase units (RLU).
3
Quantifying Wnt Pathway Activity
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The wild‐type (TOP) and mutant‐type (FOP) LEF/TCF reporter genes were cloned to the pGL3 luciferase constructs (Promega Corp., Madison, WI, USA). Cells in each group were cultured in 48‐well plates at a density of 2 × 104 cells per well for 24 h. Each well was loaded with 0.2 μg TOP/FOP flash and 1 ng Renilla (pRLTK) luciferase‐encoding plasmid (Promega) according to the instructions of a Lipofectamine 3000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). After 24 h, the luciferase intensity and Renilla signals were detected using a dual‐luciferase reporter gene kit (Promega) according to the kit's instructions. The calculation formula was as follows: TOP‐flash/FOP‐flash (Liu et al., 2021 (link); Lu et al., 2018 (link)).
4
FASN Promoter Regulation by Trastuzumab, LY294002, and U0126
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Cells were trypsinized and re-plated in 24-well plates at a density of 50,000 cells/well. Cells were incubated for 18 h to allow for attachment and were then transfected with 300 ng/well of the pGL3-Luciferase construct (Promega, Madison, WI, USA) containing a Luciferase reporter gene driven by a 178-bp FASN promoter fragment using FuGENE 6 transfection reagent (Roche Biochemicals). An internal control plasmid pRL-CMV (30 ng/well) was used to correct for transfection efficiency. After 18 h, transfected cells were washed and then incubated with trastuzumab, LY294002, or U0126 as specified, or vehicles as controls. Approximately 24 h after treatments, Luciferase assays were performed as previously described [19 (link),20 (link)].
5
DHRS9 Promoter Luciferase Assay
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Genomic DNA containing the human DHRS9 promoter regions −1989/−950 and −1061/+451 were cloned into pGL3-luciferase construct (Promega, Madison, WI, USA). Luciferase assays were performed using the Dual Luciferase system (Promega). HCT116 cells were plated at a density of 3 × 105 cells per well of a 12-well plate and transfected with a mixture containing luciferase reporter plasmid, HAND1 expression vector. Forty-eight hours after transfection, the luciferase activities were analyzed using the Dual Luciferase system.
6
Investigating miR-34a-5p Binding in Glioma
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CircITGA7-WT, circITGA7-MUT, and VEGFA-3′UTR-wide-type (WT), VEGFA-3′UTR- mutant-type (MUT) were constructed. MiR-34a-5p binding sites inserted into circITGA7 and VEGFA 3′UTR WT or (MUT were separately cloned into pGL3 luciferase construct (Promega, WI, USA). MiR-34a-5p mimics with luciferase reporter constructs and pRL-TK vector (Promega) were individually transfected into glioma cells. Relative luciferase activity was determined as manufacturers described at 24 h post-transfection.
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