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. 2017 Oct 10;8:818. doi: 10.1038/s41467-017-00932-7

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© The Author(s) 2017

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. 3 — Increased stabilization and activation of mutant NRF2. a Representative western blot of endogenous level of NRF2, KEAP1, G6PD, AKR1B10 and AKR1C1 in protein lysates of human primary fibroblast cell lines from two controls (NRF2 WT 1, WT 2) and patient 1 with NRF2 p.T80K variant. Full blots are shown in Supplementary Fig. 6. b Quantitative analysis of western blot images illustrating the endogenous level of NRF2, KEAP1, G6PD, AKR1B10 and AKR1C1 relative normalized to ACTB and NRF2 WT 1. c qRT–PCR analysis of NFE2L2, KEAP1 and target gene expression in primary fibroblast cell lines from two controls (NRF2 WT 1, WT 2) and patient 1 with NRF2 p.T80K variant. AKR1B10 and AKR1C1 are visualized on a separated X axis due to the high range. Expression is normalized to that of ACTB. % of mRNA is equal to 2^−∆∆CT and normalized relative to NRF2 WT 1. Redox calibration confirms full functionality of roGFP1 as well as identical response ranges for NRF2 WT 2 and NRF2 p.T80K fibroblast cells. d Response range calibration of an exemplary NRF2 WT 2 and NRF2 p.T80K fibroblast cell performed as a continuous recording of the roGFP1 ratio F₃₉₅/F₄₇₀ within a ROI of cytoplasm of the cell, scale bar is 20 µM. Plotted traces represent full oxidation (R_ox, induced by 5 mM H₂O₂, 5 min) and full reduction (R_red, induced by 10 mM DTT, 5 min). After calibration the relative degrees of roGFP1 oxidation and corresponding roGFP1 redox potentials can be calculated. e Baseline redox conditions of NRF2 WT 2 and NRF2 p.T80K fibroblasts. Upper diagram shows the relative level of roGFP1 oxidation of NRF2 WT 2 and NRF2 p.T80K cells at rest (OxD_roGFP1, Eq. 1). Lower diagram represents corresponding steady-state roGFP1 redox potential (E_roGFP1, Eq. 2). b, c Data are given as means ± SEM, n ≥ 3 independent experiments. Data were analyzed by one-way analysis of variance with multiple comparisons: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. e Data are given as means ± SEM. Number of measured cells are given within the bar. Statistical differences were obtained with unpaired Welch’s t-test: ***p ≤ 0.001