Pcdna3.1 flag nrf2

Manufactured by Addgene

PCDNA3.1 FLAG NRF2 is a plasmid vector that expresses the NRF2 (Nuclear Factor, Erythroid 2 Like 2) protein with a FLAG tag. The plasmid is designed for eukaryotic expression and contains a CMV promoter to drive the expression of the NRF2-FLAG fusion protein.

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Lab products found in correlation

Flag keap1, by Addgene (2 mentions) Pcdna3 egfp c4 nrf2, by Addgene (1 mentions) Pgl4.74 renilla luciferase, by Promega (1 mentions) Flag nrf2, by Addgene (1 mentions) Cignal antioxidant response reporter assay kit, by Qiagen (1 mentions) Dual glo luciferase kit, by Promega (1 mentions) 96 well plate, by Corning (1 mentions) Polyjet, by SignaGen (1 mentions)

Close spelling variants of this product

NC16 pCDNA3.1 FLAG NRF2 PCDNA3.1-NRF2 PcDNA3.1(+)-FLAG Flag‐NRF2 Flag-pcDNA3.1 PcDNA3.1

2 protocols using pcdna3.1 flag nrf2

Plasmids for NRF2 and HIF1α Regulation

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For transient transfections the following plasmids were used: pGL3-8xARE^{42 (link)}, or pGLHIF1.3^{43 (link)}, pGL4.74 renilla luciferase (#E6921, Promega), pGFP-NRF2 (pcDNA3-EGFP-C4-Nrf2, #21549, Addgene), Flag-NRF2 (pCDNA3.1 FLAG NRF2, #36971, Addgene), Flag-Keap1 (#28023, Addgene), p(HA)HIF1alpha (P402A,P564A) (#52636, Addgene). To generate plasmid GFP-NRF2-S40A, a modified QuickChange protocol was used^{44 (link)} to perform a PCR using pGFP-NRF2 as the template and primers NRF2-S40A-for (5ʹ-GTCGAGAAGTATTTGACTTCGCCCAGCGACGGAAA-GAGTATGAGCTGGAAAA-3ʹ) and NRF2-S40A-rev (5ʹ-GGCGAAGTCAAATAC-TTCTCGACTTACTCCAAGA TCTATATCTTGCCTCCAAAGTATGTCA-3ʹ). pLKO.1-shRNA-NRF2 tet-on (tetracycline inducible) contained the sequence 5ʹ-GCTCCTACTGTGATGTGAAAT-3ʹ of NRF2 mRNA (GenBank acc. no. NM_001145412), pLKO.1-shRNA-PERK tet-on (tetracycline inducible) contained the sequence 5ʹ-GGAACGACCTGAAGCTATAAA-3ʹ of PERK mRNA (GenBank acc. no. NM_004836).

Küper A., Baumann J., Göpelt K., Baumann M., Sänger C., Metzen E., Kranz P, & Brockmeier U. (2021). Overcoming hypoxia-induced resistance of pancreatic and lung tumor cells by disrupting the PERK-NRF2-HIF-axis. Cell Death & Disease, 12(1), 82.

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Measuring Nrf2 Activity in C33A Cells

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The Cignal Antioxidant Response reporter assay kit (Qiagen) was used to measure Nrf2 activity in C33A cells according to manufacturer’s instructions. Briefly, cells were seeded in 96-well plates (Costar) and transfected using PolyJet (SignaGen Laboratories) with the following plasmids: Cignal ARE Luciferase Reporter, Negative Control or Positive Control mix (25 ng), pcDNA4/TO-based pcHPV31-E1-S, pcGFP-SF or an empty vector as control (50 ng), pCDNA3.1 FLAG NRF2 (12.5 ng; Addgene #36971) and Flag-Keap1 (37.5 ng; Addgene #28023). Medium was replaced 6 hours post transfection and cells harvested 36-40 hours post transfection. Luciferase activity (Firefly and Renilla) was measured using the Dual-Glo-Luciferase kit (Promega) according to manufacturer’s instructions, and Nrf2 (Firefly) activity calculated by normalization to the Renilla luciferase readings.

Eckhardt M., Zhang W., Gross A.M., Von Dollen J., Johnson J.R., Franks-Skiba K.E., Swaney D.L., Johnson T.L., Jang G.M., Shah P.S., Brand T.M., Archambault J., Kreisberg J.F., Grandis J.R., Ideker T, & Krogan N.J. (2018). Multiple Routes to Oncogenesis are Promoted by the Human Papillomavirus-Host Protein Network. Cancer discovery, 8(11), 1474-1489.

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