The ARE-luciferase reporter construct (ARE-luc) contains the core sequence of human NQO1-ARE [35 (link)]. The mock plasmid (pcDNA3-mRFP) was purchased from Addgene (plasmid 13032). The following plasmids were previously reported. The Keap1-CBD plasmid carries the entire open reading frame (ORF) of human Keap1 fused to the chitin binding domain (CBD) of the Bacillus circulans chitinase A1 gene upstream of the Keap1 stop codon [36 (link)]. Oligonucleotide-directed mutagenesis was used to generate the Keap1-C151S-CBD plasmids. The HA-Cul3 plasmid was constructed by fusing an N-terminal hemagglutinin (HA) tag with a cDNA sequence coding for amino acids 1‒380 of human Cul3 gene. The Gal4-Neh2 expression vector contains the codons for the first 97 amino acids (Neh2 domain) of human Nrf2 fused to the ORF of the Gal4 DNA-binding domain.
Pcdna3 mrfp
Manufactured by Addgene
The pcDNA3-mRFP is a plasmid vector that contains the coding sequence for the monomeric red fluorescent protein (mRFP) under the control of the cytomegalovirus (CMV) promoter. It can be used for the expression of mRFP in mammalian cell lines.
Automatically generated - may contain errors
4 protocols using pcdna3 mrfp
1
Plasmid Constructs for ARE-luciferase Assay
2
Adenoviral Vector Construction via Homologous Recombination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RFP gene was subcloned from NruI, XbaI-digested pcDNA3-mRFP (Addgene, #13032) into the adenoviral E1 region shuttle vector SnaBI, XbaI-digested pCA14. The resulting adenoviral shuttle vector, pCA14-RFP, was linearized by XmnI digestion. The adenoviral vector dl324-BstBI was linearized by Bsp119I digestion, and the two linearized vectors were cotransformed into E. coli BJ5183 competent cells for homologous recombination.
3
Plasmid Cloning and Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pBJ-FLAG-hPar4 (cat #53231), pCMVHA hEZH2 (cat #24230), pcDNA3-mRFP (cat #13032), pCMV-VSV-G (cat #8454) and pCMV-dR8.2 dvpr (cat #8455) plasmids were purchased from Addgene. Human PAR2 (hPar2/ f2rl1) plasmid was kindly provided by Dr. Morley D. Hollenberg (Faculty of Medicine, University of Calgary, Calgary, AB, Canada). flg-β-catenin and flg-Axin were kindly provided by Dr. Ben-Neriah (Hebrew University, Jerusalem, Israel). All of the mentioned plasmids were sequenced to confirm the absence of undesirable mutations. Details of the plasmids are available on request.
4
Cloning and Transfection of CD25 Mutant Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cd25 cDNA from WT and CD25Y129H mice was amplified by using the following primers: forward 5′-CATCATGGATCCATGGAGCCACGCTTGCTGATGT-3′ and reverse 5′-CATCATGAATTCGATGGTTCTTCTGCTCTTCC-3′, which inserted BamHI and EcoRI restriction sites into the 5′ and 3′ ends, respectively. The PCR product was digested with BamHI and EcoRI restriction enzymes and subcloned into pcDNA3-mRFP (Addgene plasmid #13032). A total of 5 × 106 293T cells per 10-cm dish were seeded and transfected the next day using a calcium phosphate transfection kit (Sigma) with 10 μg plasmid. Transfected cells were cultured for 24 h in complete DMEM supplemented with 20 mM HEPES (Sigma-Aldrich) at 37 °C with 5% CO2 before FACS analysis.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!