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. 2020 Aug 25;9(9):783. doi: 10.3390/antiox9090783

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Effects of HA on NRF2 ubiquitination and E3 ubiquitin ligase in HK-2 cells. (A) Levels of NRF2 ubiquitination in cells pretreated with 10 μM MG132 (a proteasome inhibitor) or the vehicle, followed by treatment with 1000 μM HA. (B) Relative mRNA and (C) protein expression levels of Cullin 3 (CUL3) and Kelch-like ECH-associated protein 1 (KEAP1) after treatment with 0–1000 μM HA. Quantification was performed by densitometry. (D) Coimmunoprecipitation analysis of interactions between KEAP1 and CUL3 or NRF2 in cells treated with 1000 μM HA or the vehicle. (E) Surface representation of KEAP1 with a bound HA molecule (shown in a ball and stick format). (F) HA-binding site of KEAP1, shown with surface rendering. HA is depicted in a ball and stick format. (G) 2D detailed schematic of interactions between HA and KEAP1. Quantitative data are presented as the mean ± standard deviation. Experiments were repeated three to four times.