The FLAG-NRF2 expression construct was obtained from Addgene (NC16 pCDNA3.1 FLAG NRF2, Plasmid #36971, NM_006164.5), and a site-directed mutagenesis kit (Q5 Site-Directed Mutagenesis Kit, NEB, Ipswich, MA, USA) was used to introduce the T80D mutation. Codon-optimized sequences encoding truncated, FLAG-tagged transgenes of NRF1 (NM_003204.3) and NRF3 (NM_004289.7) were obtained from Integrated DNA Technologies as gBlock HiFi Gene Fragments and cloned into the pCDNA3.1 backbone from FLAG-NRF2 via Gibson assembly using a HiFi DNA Assembly Cloning Kit (NEB).
Nc16 pcdna3.1 flag nrf2
Manufactured by Addgene
Sourced in United States
The NC16 pCDNA3.1 FLAG NRF2 is a plasmid vector that expresses the transcription factor NRF2 with a FLAG tag. The plasmid is designed for use in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Hifi dna assembly cloning kit, by New England Biolabs (1 mentions)
Gblock hifi gene fragments, by New England Biolabs (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 empty vector, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Prk atf4, by Addgene (1 mentions)
3 protocols using nc16 pcdna3.1 flag nrf2
1
Generating FLAG-tagged NRF Transcription Factors
2
NRF2 Overexpression in HeLa Cells
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HeLa cells were plated at a density of 30% in six-well plates and the transfection procedure was performed the following day using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA, 15338-100) according to the manufacturer’s indications in Opti-MEM (Thermo Fisher Scientific, 11058021). One microgram of NC16 pcDNA3.1 Flag NRF2 (Addgene, Watertown, MA, USA, 36971) or the corresponding pcDNA3.1 empty vector (Invitrogen) was transfected into each well. After 4 h of transfection, complete DMEM was added to each well. Cellular extracts were collected after 24 h of plasmid transfection.
3
Plasmid Constructs for UPR Study
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The expression vector for pBMN-I-GFP, pBMN-hATF6(373)-I-GFP encoding aa 1–373 of human ATF6α and pBMN-hXBP1(S)-I-GFP encoding full-length human XBP1 generated by UPR-mediated splicing were a kind gift from Dr Joseph Brewer (University of South Alabama, Mobile, AL, USA). The expression vector for FLAG-tagged ATF4 (pRK-ATF4) was a gift from Yihong Ye (Addgene plasmid # 26114; Cambridge, MA, USA); FLAG-tagged NRF2 (NC16 pCDNA3.1-FLAG-NRF2) was a gift from Randall Moon (Addgene plasmid # 36971). The expression vector for FLAG-tagged CHOP (pcDNA3-FLAG-CHOP) was a kind gift from Dr Wolfgang Dubiel (Humboldt University, Germany). The pGL3-ACTR-1.6 kb construct was a kind gift from Dr Hongwu Chen (University of California, Davis, CA, USA) and contains a genomic DNA fragment (1.6 kb, HindIII–NcoI) containing the first exon of NCOA3 into vector pGL3-basic. The ATF6, PERK and IRE1–XBP1 pathway reporters has been described previously.46 (link)
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