Cells were co-transfected with shLacZ, shBCL9, shILF2 plasmids, and subsequently transfected with NFAT luciferase reporter (Plasmid #10959, Addgene) and SV40 drive renilla luciferase reporter (E2231, Promega). 24 h post-transfection, 1 × 105 cells were plated per well in 96-well plates. 24 h after seeding, the Dual-Luciferase Reporter Assay System (Promega) was used to measure luminescence according to the manufacturer’s instructions. The luciferase signal from the NFAT reporter was normalized to the luciferase signal from the renilla reporter.
E2231
Manufactured by Promega
Sourced in United States
E2231 is a protein purification reagent designed for the isolation of recombinant proteins expressed in E. coli. It can be used to purify proteins tagged with a hexahistidine (His-tag) sequence.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
E1751, by Promega (1 mentions)
Mutanbest kit, by Takara Bio (1 mentions)
E1980, by Promega (1 mentions)
E1910, by Promega (1 mentions)
Luminometer, by Agilent Technologies (1 mentions)
7 protocols using e2231
1
NFAT Luciferase Reporter Assay
2
Evaluating NF-κB Transactivation and miRNA Targets
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To assess the transactivation of NF-κB, a pGL4.32 [luc2P/NF-κB-RE/Hygro] vector (E849A; Promega, Madison, WI, USA) containing an NF-κB responsive element was used. U2OS cells were transfected with siRNA or miRNA for 24 h and then the firefly reporter vectors were transfected along with renilla expressing vector (E2231; Promega).
For validation of miRNA recognition elements, the luciferase constructs containing the binding sites of miR-1908-5p were manufactured using a pmirGLO vector (E133A; Promega). Since two positions are predicted for the miR-1908-5p binding site in the 3’UTR of NKIRAS2 mRNA, we prepared two individual vectors containing wild-type or mutant sequences of miR-1908-5p. In order to check the effect of miR-1908-5p on NKIRAS2 expression, U2OS cells were seeded in 24-well plates with around 50% confluency and transfected with the indicated siRNA or miRNA. At 24 h post-transfection, cells were transfected with either a pmirGLO blank vector or pmirGLO-NKIRAS2/3’UTR vector (willd-type or mutant). The luciferase activity was measured 24 h later using Dual-Glo Luciferase Assay System (E2940; Promega) on a GloMax®20/20 Luminometer (Promega).
For validation of miRNA recognition elements, the luciferase constructs containing the binding sites of miR-1908-5p were manufactured using a pmirGLO vector (E133A; Promega). Since two positions are predicted for the miR-1908-5p binding site in the 3’UTR of NKIRAS2 mRNA, we prepared two individual vectors containing wild-type or mutant sequences of miR-1908-5p. In order to check the effect of miR-1908-5p on NKIRAS2 expression, U2OS cells were seeded in 24-well plates with around 50% confluency and transfected with the indicated siRNA or miRNA. At 24 h post-transfection, cells were transfected with either a pmirGLO blank vector or pmirGLO-NKIRAS2/3’UTR vector (willd-type or mutant). The luciferase activity was measured 24 h later using Dual-Glo Luciferase Assay System (E2940; Promega) on a GloMax®20/20 Luminometer (Promega).
3
Cloning and Transfection of BECN1 Constructs
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As described previously [12 (link)], 3′-UTR, 5′-UTR and CDS of human BECN1 were cloned into pGL3-Luc vector (E1751, Promega). Then, pGL3-Luc-BECN1-3′UTR, pGL3-Luc-BECN1-CDS and pGL3-Luc-BECN1-5′UTR plasmids were transfected into cells. To monitor transfection efficiencies, the luciferase reporter vectors were transfected in cells together with phRL-null (E2231, Promega).
4
Characterization of CYP2J2 3'-UTR
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A human CYP2J2 3’-UTR (-2000/+100 bp) fragment was isolated from a human genomic library and cloned into the XhoI and NotI sites of the PGL-3-basic luciferase reporter vector. A series of CYP2J2-promoter deletion constructs was created and amplified by PCR. For mutations of the putative AP1/SP1–like elements, binding sites were generated from the CYP2J2-1000/+100-bp construct by using the TaKaRa MutanBEST Kit (TaKaRa Biotechnology, China). All constructs were confirmed by sequencing. Cells were co-transfected with the corresponding reporter plasmid and CMV–β-gal plasmid (to normalize for transfection efficiency) in each experiment according to the manufacturer's instructions. The dual-luciferase reporter assay (Promega, E2231) was performed after 24 h in accordance with the manufacturer's instructions.
5
Detecting HIF-1α Activity via Luciferase Assay
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HIF-1α activity was detected by transfecting The 6 × HRE luciferase vector. Methylated and unmethylated PCR products were cloned into the PGL3-enhancer vector. All constructs were confirmed by Sanger sequencing. The Renilla luciferase vector was co-transfected and used to normalize transfection efficiency using the dual-luciferase reporter assay kit (E1980, Promega, Madison, WI, USA) according to the manufacturer’s instructions. All data were normalized to Renilla luciferase luminescence derived from the co-transfected pRL-SV40 vector (E2231, Promega, Madison, WI, USA).
6
Regulation of Mir181a2b2 Promoter Activity
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HEK 293 T cells were plated on 12-well plates at 300,000/well up to 70–80% confluency and transfected with 500 ng of the indicated Mir181a2b2 promoter reporter constructs (Supplementary file 1) and 10 ng Renilla plasmid (E2231, Promega). Adenosine receptor siRNAs and SMAD2/3/4 siRNAs were co-transfected at 100 or 20 nM final concentration and cells were treated with MTX (10 µM) or Adenosine (50 µM) 24 hr post-transfection for 12 hr. Transfected cells were collected in 200 µl reporter lysis buffer (E1910, Promega) and luciferase activity was measured using a Dual-Luciferase reporter assay system (E1910, Promega). Each reading of luciferase activity was normalized to the Renilla activity.
7
Regulation of Nix by PHB1 in Rotenone-Treated Cells
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Mode-K cells were co-transfected with pGL3 or pGL3-Nix, siNC or siPHB1, and pRL-CMV (Renilla luciferase as internal control; E2231, Promega). 72 h post-transfection, cells treated with 500 nM rotenone for 2 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (E1910, Promega) and a BioTek luminometer. Relative luciferase was calculated by normalizing firefly luciferase activity to renilla luciferase activity.
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