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. 2020 May 18;11:2476. doi: 10.1038/s41467-020-16129-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Relative to Chicken (Gallus gallus; [N = 6 biologically independent animals]), liver NRF2 mRNA expression is significantly upregulated in wild Neoaves species (White-throated sparrow Zonotrichia albicollis [N = 3 biologically independent animals; p = 0.014]; American woodcock Scolopax minor [N = 2 biologically independent animals; p = 4.67 × 10⁻⁷]; one-sided t test). All data are presented as mean values. b NRF2 protein levels are elevated in both the cytoplasmic and nuclear fractions of wild Neoaves liver lysates (Oven bird Seiurus aurocapilla [N = 2 biologically independent animals]; Black-billed cuckoo Coccyzus erythropthalmus [N = 1 biologically independent animal]) relative to Chicken (Gallus gallus; [N = 2 biologically independent animals]). p = 0.0078 and 0.036 for cytoplasmic and nuclear, respectively. One-sided t test. Consistent with dysregulation of NRF2 by KEAP1, Neoaves (Oven bird Seiurus aurocapilla; [N = 2 biologically independent animals]) display increased nuclear to cytoplasmic ratios, relative to Chicken [N = 3 biologically independent animals]. p = 0.038, one-sided t test. c Liver chromatin immunoprecipitation using an anti-NRF2 antibody reveals that Chicken and Neoaves (Oven bird Seiurus aurocapilla; Gray catbird Dumetella carolinensis) NRF2 is bound to antioxidant-response elements (ARE) of GCLC and PRDX1 in vivo. NRF2 AREs could not be detected in Neoaves genomes, yet two AREs are bound by NRF2 in Chicken liver (note that IgG background is undetectable for all AREs, except for one of the two NRF2 AREs identified in Chicken, where it comprises only 0.6% of signal obtained with the NRF2 antibody; Source data). d NRF2 target genes (GCLC, GCLM, PRDX1, TXNRD1) are expressed in wild Neoaves (White-throated sparrow Zonotrichia albicollis [N = 3 biologically independent animals]; American woodcock Scolopax minor [N = 2 biologically independent animals]) and are significantly upregulated relative to Chicken [N = 8 biologically independent animals]. **p < 0.01; ***p < 0.001. One-sided t test. Source data are provided as a Source data file. All error bars represent standard error.