Abstract—
Reporter vectors expressing of amino acid residues 16–96 and 16–85 from Neh2-domain of Nrf2 transcription factor fused to firefly luciferase have been constructed. Cell lines stably expressing the above reporter constructs as well as an earlier developed Neh2-luc fusion reporter have been generated using HeLa cells. Comparative analysis of all three reporter lines has been performed using a well-known Nrf2 activator, nordihydroguaiaretic acid. The reporter expressing the minimal fragment of Neh2-domain containing 16–85 aa sequence exhibits a 2-fold higher amplitude of activation compared to Neh2-luc reporter, and thus can be recommended for high throughput screening for Nrf2 activators. A likely reason for the improved response of the above reporter could be ubiquitination of N-terminal lysine residues (7, 10, 11) of luciferase protein adjacent to the ubiquitinated Neh2 sequence.
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The work was supported in part by Russian Foundation for Basic Research (project no. 20-04-00921).
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Abbreviations used: NDGA, nordihydroguaiaretic acid; DMSO, dimethyl sulfoxide; DMEM, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; PCR, polymerase chain reaction, HRP, horseradish peroxidase; PBS, phosphate buffer saline.
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Khristichenko, Y., Hushpulian, D.M., Smirnova, N.A. et al. Optimization of Neh2-Luc Reporter for Screening of Activators of Antioxidant Program. Moscow Univ. Chem. Bull. 75, 172–178 (2020). https://doi.org/10.3103/S0027131420030062
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DOI: https://doi.org/10.3103/S0027131420030062