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Protocol to profile spatially resolved NLRP3 inflammasome complexes using APEX2-based proximity labeling
Zhu Liang1, Andreas Damianou1, Athina Grigoriou2
1Chinese Academy for Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK; Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
None:
The NLRP3 inflammasome is a key multi-protein complex controlling inflammation, particularly interleukin-1β (IL-1β) production. Here, we present a protocol to profile spatially resolved NLRP3 inflammasome complexes using ascorbic peroxidase 2 (APEX2)-based proximity labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for design and generation of the fusion construct, characterization of the stable FLAG-NLRP3-APEX2 expression cell line by western blotting/imaging, biotinylated proteome enrichment, and mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Liang et al.1.
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