The synthetic Smad7 3'UTR gene fragment Smad7 wild type (WT) and Smad7 mutant (MUT) with binding site mutation were constructed into pMIR reporter plasmid (Beijing Huayueyang Biotechnology, Beijing, China). Luciferase reporter plasmids (smad7-WT and MUT) were cotransfected with miR-195-5p into HEK293T cells (Shanghai Beinuo Biotechnology, Shanghai, China). At 48h post transfection, the cells were lysed. Luciferase assay kit (K801-200; Biovision, Mountain View, CA, United States) was used for detection of luciferase activity.
Luciferase assay kit
Manufactured by Abcam
Sourced in United States
The Luciferase Assay Kit is a laboratory tool used to measure the activity of the luciferase enzyme. Luciferase is a protein found in various organisms that emits light through a bioluminescent reaction. The kit provides the necessary reagents and protocols to quantify luciferase activity, which can be used as a reporter for gene expression or other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Glomax 20 20 luminometer, by Promega (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Pmir reporter plasmid, by Huayueyang (3 mentions)
Ag sybr green pro taqhs premix, by Accurate Biology (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Glomax20 20 luminometer fluorescence detector, by Promega (2 mentions)
Plasmid extraction kit, by Promega (2 mentions)
293t cell, by American Type Culture Collection (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 kit, by Thermo Fisher Scientific (1 mentions)
Pmir report reporter, by Thermo Fisher Scientific (1 mentions)
Td 20 20 luminometer, by Promega (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
Pgl3 mut jmjd2c 3 utr, by GenePharma (1 mentions)
Tgf β, by Proteintech (1 mentions)
Dual luciferase reporter assay reagent, by GeneCopoeia (1 mentions)
Lps l2880, by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit 1 556 547, by BD (1 mentions)
Dual luciferase reporter gene analysis system, by Promega (1 mentions)
Phospho p38 mapk, by Abcam (1 mentions)
41 protocols using luciferase assay kit
1
Dissecting miR-195-5p Regulation of Smad7
2
Luciferase Assay for TPX2 3' UTR Interaction
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Synthetic TPX2 3′ UTR gene fragment was incorporated into a pMIR-reporter plasmid (Huayueyang Biotechnology, Beijing, China). The complementary sequence mutation sites were designed based on the TPX2-WT sequence. Using restriction endonuclease digestions and ligations with the T4 DNA ligase, the target fragments were inserted into the pMIR-reporter plasmid, which were later co-transfected into HEK293T cells (Beinuo Biotechnology, Shanghai, China) using the miR-216b mimic. Luciferase activity was detected using a luciferase assay kit (K801-200, BioVision, Mountain View, CA, USA).
3
Molecular Profiling of Apoptotic Pathways
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The antibody GRK4 (Cat. No. 11808-1-AP) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved caspase−3 (Cat. No. 9661s) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Caspase-3 (Cat. No. ab184787) was obtained from Abcam (Cambridge, MA, USA). Anti-β-Tubulin (Cat. No. T0023) was purchased from Affinity Biosciences (Cincinnati, OH, USA). The luciferase assay kit was obtained from BioVision (Milpitas, CA, USA). The lncRNA 148400 siRNA, miR−10b−3p mimic, miR−10b−3p inhibitor, GRK4 siRNA, and GRK4 plasmid were purchased from Ribo (Guangzhou, China). Lipofectamine 2000 was obtained from Life Technologies (Carlsbad, CA, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). The Ag SYBR Green Pro TaqHS premix was obtained from Accurate Biotechnology (China).
4
Validating miR-143-3p Target Genes
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The biological prediction website microRNA.org was used for analysis of the target gene of miR-143-3p and dual-luciferase reporter gene assay was conducted to verify whether JAG1/NOTCH2 was a direct target gene of miR-143-3p. The constructed JAG1/NOTCH2-wild type (WT) and JAG1/NOTCH2-mutant (MUT) were co-transfected with miR-143-3p mimic or mimic NC to HEK-293T cells (Shanghai BeiNuo Biotechnology Co., Ltd., Shanghai, China). After 48-h transfection, the cells were collected and lysed, and luciferase activity was determined by means of luciferase assay kit (K801-200, Biovision, Milpitas, CA, USA) using the glomax20/20 luminometer (Promega WI, USA).
The miR-143-3p promoter region was cloned into the Luciferase vector of pmirGLO (Promega) to construct the miR-143-3p Promoter WT (wild-type miR-143-3p Promoter plasmid), miR-143-3p Promoter mut1 (miR-143-3p Promoter plasmid at mutation site 1) and miR-143-3p Promoter mut2 (miR-143-3p Promoter plasmid at mutation site 2). The HEK-293T cells were seeded into a 24-well plate. After 24 h, 100 ng oe-NC or 100 ng oe-GR were co-transfected with 50 nmol/L miR-143-3p Promoter plasmid according to Lipofectamine 2000 kit (Invitrogen), and the luciferase activity was measured 48 h after transfection.
The miR-143-3p promoter region was cloned into the Luciferase vector of pmirGLO (Promega) to construct the miR-143-3p Promoter WT (wild-type miR-143-3p Promoter plasmid), miR-143-3p Promoter mut1 (miR-143-3p Promoter plasmid at mutation site 1) and miR-143-3p Promoter mut2 (miR-143-3p Promoter plasmid at mutation site 2). The HEK-293T cells were seeded into a 24-well plate. After 24 h, 100 ng oe-NC or 100 ng oe-GR were co-transfected with 50 nmol/L miR-143-3p Promoter plasmid according to Lipofectamine 2000 kit (Invitrogen), and the luciferase activity was measured 48 h after transfection.
5
Validating miR-615-5p Binding Sites
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The binding sites between miR-615-5p and circ-OGDH or PDX1 were predicted using circular RNA interactome and TargetScan. The fragments of wild type (wt) circ-OGDH and 3ʹ untranslated regions (UTR) of PDX1 and their mutant (mut) sequences were inserted into the downstream of the pMIR-REPORT reporter (Applied Biosystems), respectively. ESCC cells were co-transfected with a luciferase reporter containing wt-circ-OGDH, mut-circ-OGDH, wt-PDX1 3ʹUTR, or mut-PDX1 3ʹUTR and miR-NC or miR-615-5p mimic. Thereafter, the cells were lysed and the luciferase activities were assessed with a luciferase assay kit (Biovision) in a TD20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA).
6
JMJD2C 3'UTR Luciferase Reporter Assay
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Wild type (wt) (pGL3-wt-JMJD2C-3’untranslated region [UTR]) or mutant (mut) (pGL3-mut-JMJD2C-3’UTR) reporter plasmids (GenePharma, Shanghai, China) were co-transfected with agomir NC or miR-216b agomir into MG63 and SaOS-2 cells. Following a 48 h period of transfection, the cells were collected and lysed. The subsequent procedures were performed with using a luciferase assay kit (K801–200, Biovision, Milpitas, CA, USA) following the manufacturer’s instructions on the luciferase reporter assay system (Promega, Madison, WI, USA). The relative luciferase activity was calculated based on the ratio of the luciferase activity of firefly luciferase to that of renilla luciferase. The sequences of wt-JMJD2C-3’UTR and mut-JMJD2C-3’UTR were 5′-GCAUGUAUGCUAAUGAGAUUU-3′ and 5′-GCUGUAAACGACGUCUCUAAA-3′, respectively [30 (link)].
7
Molecular Signaling in Tissue Repair
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Primary antibodies against p38 MAPK (Cat. No. ab31828), collagen I (Cat. No. ab34710), collagen III (Cat. No. ab7778), fibronectin (Cat. No.ab2413), HBEGF (Cat. No. ab92620), and KRAS (Cat. no. ab180772) were purchased from Abcam (Cambridge, MA, USA). Anti- GAPDH antibody (Cat. No. 10494-1-AP) and TGF-β were obtained from Proteintech North America (Rosemont, IL, USA). Anti-phospho-p38MAPK antibody (Cat. No. 4511) was purchased from Cell Signaling Technology (Danvers, MA, USA). The luciferase assay kit was purchased from BioVision (Milpitas, CA, USA). All plasmids used in this study were generated by Vigene Biosciences (Jinan, Shangdong, China). The p38 MAPK inhibitor, SB203580 (Cat. No. HY-10256) and p38MAPK agonist (Cat. #HY-N0674A/CS-6061) were purchased from MedChemExpress USA (Deer Park, NJ, USA).
8
Antibody Selection and Validation Protocol
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Antibody Rab10 (Cat. No. 11808-1-AP) and Antibody p53 (Cat. No.60283-2-Ig) were obtained from ProteinTech North America (Rosemont, IL, USA). Anti-cleaved caspase-3 (Cat. No. 9661s) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase-3 (Cat. No. ab184787) was obtained from Abcam (Cambridge, MA, USA). Anti-β-tubulin (Cat. No. T0023) was purchased from Affinity Biosciences (Cincinnati, OH, USA). Anti-bax (Cat. No.AF120) and bcl2 (Cat. No. AF6139) was purchased from Affinity Biosciences (Cincinnati, OH, USA). The luciferase assay kit was obtained from BioVision (Milpitas, CA, USA). Anti-β-Tubulin (Cat. No. 86298) was purchased from Cell Signaling Technology (Danvers, MA, USA).
9
Validating miR-140-5p Regulation of BMP2
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For the purpose of verifying the binding relationship between miR-140-5p and BMP2, and to assess whether BMP2 is a direct target gene of miR-140-5p, ORSCs were seeded in 6-well plates (Guangzhou Jet Bio-Filtration Co., Ltd, TCP-010-006) at a density of 2.5 × 105 cells/well with the complete media. The artificially synthesized 3′-UTR of BMP2 was inserted into the pMIR-reporter vector (Yue Yang Biotechnology Co., Ltd., Beijing, China). A site-specific mutation was then introduced into the 3′-UTR fragment. One microgram of BMP2-wild type (WT) or BMP2-mutant type (MUT) was cotransfected into ORSCs with 50 nM miR-140-5p mimic or miR-NC. Transfections were performed using Lipofectamine 2000 (Invitrogen) with Opti-MEM (Thermo Fisher Scientific). After 48 h, the cells were lysed and subjected to luciferase assay using the dual-luciferase reporter assay reagent (GeneCopoeia, Rockville, MD, United States). Luciferase activity was detected using the Luciferase Assay kit (K801-200; Biovision, San Francisco, CA, United States) on a Glomax20/20 luminometer (Promega Corporation, Madison, WI, United States). The 3′-UTR of BMP2 was obtained from GeneCopoeia.
10
Antibody-Mediated Apoptosis Analysis
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Anti-PAK7 and anti-β-tubulin antibodies were procured from Proteintech (USA). Anti-C3 (9662) and CC3 (9664) antibodies were supplied by Cell Signaling Technology (USA). Secondary antibodies were obtained from Affinity (USA). FITC-Annexin-V-Apoptosis-Detection-Kit-I (556,547; BD Pharmingen, USA); luciferase-assay-kit (BioVision, USA); AG-SYBR-Green-Pro-Taqhs-premix (Accurate Biotechnology, China); LPS (L2880; Sigma, USA).
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