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Subcellular localization and cytoplasmic complex status of endogenous Keap1
Yoriko Watai1, Akira Kobayashi, Hiroko Nagase
1Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan.
Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|October 2, 2007
Summary
A new antibody reveals Kelch-like ECH-associated protein 1 (Keap1) primarily resides in the cytoplasm. Oxidative stress does not alter Keap1
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Keap1 regulates Nrf2 activity, a key transcription factor for antioxidant response.
- The mechanism of Nrf2 nuclear translocation under stress is unclear due to challenges in detecting endogenous Keap1.
Purpose of the Study:
- To generate a reliable monoclonal antibody for detecting endogenous Keap1.
- To investigate the subcellular localization of Keap1 under various conditions.
Main Methods:
- Monoclonal antibody generation and validation.
- Immunocytochemistry in mouse embryonic fibroblasts.
- Subcellular fractionation of mouse liver cells.
- Sucrose density gradient centrifugation.
Main Results:
- A novel antibody successfully detected endogenous Keap1.
- Keap1 is predominantly localized in the cytoplasm, with minimal nuclear or ER presence.
- Subcellular Keap1 distribution remained unchanged after electrophile or nuclear export inhibitor treatment.
- Keap1 forms cytoplasmic multiprotein complexes.
Conclusions:
- Electrophiles induce Nrf2 nuclear accumulation independently of Keap1 redistribution.
- Endogenous Keap1 primarily functions from the cytoplasm.
- The generated antibody is a valuable tool for Keap1 research.
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