Fig. 3: USP25 interacts with  KEAP1. | Nature Communications

Fig. 3: USP25 interacts with  KEAP1.

From: USP25 regulates KEAP1-NRF2 anti-oxidation axis and its inactivation protects acetaminophen-induced liver injury in male mice

Fig. 3: USP25 interacts with  KEAP1.The alternative text for this image may have been generated using AI.

a The identification of potential substrates of USP25 by mass spectrometry. FLAG-tagged USP25 was overexpressed in HEK293T cells, immunoprecipitated, separated with SDS-PAGE. The gel was stained with Commassie blue. b Proteins in the gel of (a) were identified with mass-spectrometry. Top 10 most abundant proteins identified were listed. c In vitro GST pull-down analysis of the interaction between KEAP1 (FLAG- KEAP1) and USP25 (GST-USP25). Arrows indicate the Coomassie blue staining of GST and GST-USP25. d Immunoprecipitation analysis of the interaction between FLAG-USP25 and endogenous KEAP1 as well as FLAG- KEAP1 and endogenous USP25 in HEK293T cells (n = 3 biologically independent experiments). e Co-immunoprecipitation analysis of the interaction between USP25 and KEAP1 in the liver from WT mice treated with or without APAP (300 mg/kg, i.p.) for 6 h (n = 3 biologically independent experiments). f Mapping the domains in KEAP1 that interact with USP25. FLAG-tagged BTB, IVR, and DGR domains of KEAP1 were expressed in HEK293T cells and immunoprecipitated. The immunoprecipitated were then analyzed for the presence of USP25 (n = 2 biologically independent experiments). g Mapping the domains in USP25 that interact with KEAP1. FLAG-tagged SIM/UIM, USP, and CTD domains of USP25 were expressed in HEK293T cells and immunoprecipitated. The immunoprecipitated were then analyzed for the presence of KEAP1 (n = 2 biologically independent experiments). Source data are provided as a Source Data file.

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