Reporter NRF2(ARE)-LUC was purchased from the Signosis Company (Cat# LR-2106, CA, USA). HeLa cells (5 × 104 cells/well) were plated in 24-well plates, and 1.0 μg of reporter were transfected using jetPEI (Polyplus Transfection Inc., NY, USA) following the manufacturer's protocol (Promega Luciferase Assay Kit and DLR2 model). Cells were harvested for luciferase reporter assays using a Promega Luciferase Assay Kit as described previously (26 (link)). Luciferase activities in extracts from the transfected cells are presented in terms of relative light units (RLU) and expressed as the mean and standard deviation of three transfected cultures.
Luciferase assay kit
Manufactured by Promega
Sourced in United States, China, Germany, Japan, Australia, United Kingdom
The Luciferase Assay Kit is a lab equipment product that measures the activity of luciferase enzymes. It provides the necessary reagents to quantify the amount of light produced by the luciferase-catalyzed reaction.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (144 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (54 mentions)
β galactosidase β gal expression plasmid, by Thermo Fisher Scientific (28 mentions)
Reporter lysis buffer, by Promega (28 mentions)
P mir reporter plasmid, by Thermo Fisher Scientific (25 mentions)
Pmir report plasmid, by Thermo Fisher Scientific (25 mentions)
β galactosidase expression vector, by Thermo Fisher Scientific (23 mentions)
Dual luciferase reporter assay system, by Promega (18 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (17 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
Passive lysis buffer (plb), by Promega (16 mentions)
Opti mem, by Thermo Fisher Scientific (16 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Lipofectamine, by Thermo Fisher Scientific (12 mentions)
Pmirglo vector, by Promega (12 mentions)
Pgl3 vector, by Promega (12 mentions)
Glomax luminometer, by Promega (11 mentions)
Glomax 96 microplate luminometer, by Promega (10 mentions)
Psicheck 2 vector, by Promega (10 mentions)
Pgl3 basic vector, by Promega (9 mentions)
1 063 protocols using luciferase assay kit
1
Luciferase Reporter Assay for NRF2(ARE) Activation
2
Assessing Glucocorticoid Receptor Activity
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The expression pKSX vectors for human GR and reporter gene for the MMTV-LUC have been described previously [16 (link)]. HK-2 cells were plated in 24-well plates and transfected using jetPEI (Polyplus Transfection Inc., New York, NY, USA) following the manufacturer’s protocol (Promega luciferase assay kit and DLR2 model). The total DNA was adjusted to 1.0 μg using empty pSG5.HA vector. Cells were harvested for luciferase reporter assays using a Promega luciferase assay kit (Madison, WI, USA). Values are expressed numerically as relative light units. Luciferase activity is presented as the mean ± SD of two transfected wells and is representative of at least three independent experiments. The results shown are representative of at least three independent experiments.
3
Transcriptional Regulation of DEC1 Gene
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DEC1 was constructed by inserting its full-length PCR (polymerase chain reaction) fragment into the pCMV.Flag vector via the SalI-EcoRI restriction site. pGL3. DEC1 (−940/+42)-LUC was constructed by inserting the PCR fragment into the pGL3 basic vector via the KpnI-XhoI restriction site. The expression vectors pSG5.HA.p53 fragments, pSG5.HA.mZac1, pSG5.HA.HDAC1 (wt and H141A), SV40-LT, pSG5.HA.SIRT1, and pEGFP. DEC1 were all described previously.21 (link)–25 (link) HeLa cells were plated in 24-well plates and transfected using jetPEI (Polyplus Transfection Inc., New York, NY) following the manufacturer’s protocol (Promega luciferase assay kit and DLR2 model). The total DNA was adjusted to 1.0 μg using empty vector. Cells were harvested for luciferase reporter assays using a Promega luciferase assay kit as described previously.23 (link) Luciferase activities in extracts from the transfected cells are presented in terms of relative light units (RLU) and expressed as the mean and standard deviation of three transfected cultures. Because the expression of several control vectors used to monitor transfection efficiency may be enhanced by Zac1,23 (link) internal controls were not used. Instead, the reproducibility of observed effects was determined through multiple independent transfection experiments.
4
Exosome-mediated miRNA Regulation Assay
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For luciferase reporter assays of exosomes, synthetic DNA fragments corresponding to the complementary sequences of miR156a, miR168a or miR2911 were ligated to the 3′-UTR of the pMIR-REPORT plasmid (Thermo Fisher Scientific). The HEK293T cells were incubated with exosomes resuspended in medium containing 2% FBS for 8 h, washed extensively with PBS, and then transfected with 0.25 μg of firefly luciferase reporter plasmid and 0.25 μg of β-galactosidase (β-gal) control plasmid (Thermo Fisher Scientific) per well in 24-well plates. At 10 h post transfection, cells were analyzed using a luciferase assay kit (Promega). For validation of miR2911 targeting TGF-β1, a 729-bp segment of the 3′-UTR of human TGF-β1 that contained the presumed miR2911 binding site was generated and inserted into the pMIR-REPORT plasmid. To test the binding specificity, another 529-bp segment without the binding site was generated as the mutant control. Briefly, 0.25 μg of firefly luciferase reporter plasmid, 0.25 μg of β-gal control plasmid, and 50 pmol of miR2911 were transfected into cells per well in 24-well plates for 6 h. At 24 h post transfection, the cells were assayed using a luciferase assay kit (Promega).
5
Luciferase Assay of HeLa Cell Transfection
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HeLa cells were plated in 24-well plates and transfected using jetPEI (Polyplus Transfection Inc., New York, NY, USA) following the manufacturer’s protocol (Promega luciferase assay kit and DLR2 model). The total DNA was adjusted to 1.0 μg using empty vector. Cells were harvested for luciferase reporter assays using a Promega luciferase assay kit. Values are expressed numerically as relative light units. Luciferase activity is presented as the mean ± SD of three transfected wells and is representative of at least three independent experiments.
6
TRMP Regulation by p53 and IRES Activity
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To investigate whether TRMP is transcriptionally regulated by p53, H1299 cells were co-transfected with control vector, Flag-p53, or together with the pGL3-based construct containing wild-type or mutant TRMP promoter plus Renilla luciferase plasmid in the indicated combination. Twenty-four hours later, the reporter activity was measured by using a luciferase assay kit (Promega) and plotted after normalizing with respect to Renilla luciferase activity (mean ± SD).
To determine the effect of TRMP on the IRES activity of p27 5′-UTR, HEK293T cells expressing control shRNA or TRMP shRNA were transfected with the luciferase reporter construct containing p27 5′-UTR (−447 to −1). Twenty-four hours later, the reporter activity was measured by using a luciferase assay kit (Promega) and plotted after normalizing with respect to Renilla luciferase activity. The data are shown as mean ± SD of three independent experiments.
To determine the effect of TRMP on the IRES activity of p27 5′-UTR, HEK293T cells expressing control shRNA or TRMP shRNA were transfected with the luciferase reporter construct containing p27 5′-UTR (−447 to −1). Twenty-four hours later, the reporter activity was measured by using a luciferase assay kit (Promega) and plotted after normalizing with respect to Renilla luciferase activity. The data are shown as mean ± SD of three independent experiments.
7
Luciferase Reporting Assay for miRNA Targets
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To construct luciferase reporter vectors, HOXB13 3΄-UTR and CCAT1 cDNA fragment containing the predicted micro-RNAs binding sites were amplified by PCR, and then subcloned downstream of the luciferase gene in the pGL3 plasmid. Mutant of plasmids (pGL3-HOXB13-3΄UTR-MUT and pGL3-CCAT1-MUT) were generated by site directed mutagenesis PCR reaction using platinum pfx DNA polymerase following the product manual. All constructs were verified by DNA sequencing. The luciferase assays were performed using a luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Briefly, cells were first transfected with appropriate plasmids in 24-well plates. Next, the cells were collected and lysed for luciferase assay 48 h after transfection. The relative luciferase activity was normalized with renilla luciferase activity.
8
Regulation of AP-1 Activity in BV2 Cells
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BV2 cells were plated on 24-well plate and incubated for 16 h at 37 °C. Then the cells were transfected with Cignal lentiviral AP-1 Reporter (luc) (QIAGEN, Hilden, Japan) according to the manufacturer's instructions for 24 h. Subsequently, the culture medium were replaced with fresh medium. After another 24 h, the cells were cultured in culture medium supplement with 2.5 μg/ml of puromycin. After 5 days, survived BV2 cells were selected and the BV2 stable cell line expressing AP-1 reporter construct was generated. Subsequently, the BV2 cells stably expressing AP-1 constructs were transfected with siRNA against TDP-43. After 48 h, the cells were treated with U0126 for 24 h, and then the cells were harvested. Finally, the AP-1 promoter activity was measured using the luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.
9
Validating ATG7 3'UTR-miR-138 Interaction
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The wild type (WT, containing putative miR-138-5p binding sites) or mutate (mutant, putative binding sites were mutated) 3′UTR of ATG7 were constructed into the pGL3-Enhancer Vector (Promega). The constructs were co-transfected with pRL-TK (Promega), and miR-138-5p mimics or mimics control (Con). The pRL-TK plasmid was used as a transfection control. Luciferase activity was measured using a luciferase assay kit (Promega, Madison, WI, USA) at 48 h after transfection.
10
Verification of miRNA-mRNA Interactions
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Dual luciferase reporter assays were performed to verify the direct interactions between KCNQ1OT1 and miR-34a and miR-34a and the 3′-UTR of Atg4B mRNA. PCR was conducted using the PrimeSTAR DNA polymerase (Takara, Kusatsu, Japan) to amplify the KCNQ1OT1 cDNA containing the predicted miR-34a binding site and the 3′-UTR of Atg4B cDNA containing the predicted miR-34a binding site. The primers used are presented in Table S2 . Then, the PCR products were purified and cloned to the pmirGLO vector. Afterward, we co-transfected the pmirGLO-WT-KCN1 or -Mut-KCN1 with miR-34a mimics or miRNA-NC into colon cancer cells with Lipofectamine 3000. Also, the pmirGLO-WT-Atg4B or -Mut-Atg4B was transfected in a similar way. The luciferase activity was conducted after 48 h of transfection using the luciferase assay kit (Promega Corporation, Fitchburg, WI, USA) and the Promega GloMax 20/20 machine.
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