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¹ Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan
² Yokohama Laboratory, Summit Pharmaceuticals International Corporation, Yokohama, Japan
³ Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan
⁴ Environmental Response Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, University of Tsukuba, Tsukuba, Japan
⁵ Department of Molecular Biology, Cell Biology and Biochemistry, and the Center for Genomics and Proteomics, Brown University, Providence, RI, USA
⁶ Center for Advanced Medical Research, Hirosaki University School of Medicine, Hirosaki, Japan

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

^* Correspondence: E-mail: masi{at}tara.tsukuba.ac.jp,akirak{at}mail.tains.tohoku.ac.jp

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