Protocol to profile spatially resolved NLRP3 inflammasome complexes using APEX2-based proximity labeling
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.<sup>1</sup>.
Authors
- Benedikt Mathias Kessler (ORCID: https://orcid.org/0000-0002-8160-2446)
- Iolanda Vendrell (ORCID: https://orcid.org/0000-0001-7187-4061)
- Hannah B. L. Jones (ORCID: https://orcid.org/0000-0002-7834-0625)
- Frederik Lassen
- Elena Di Daniel
- Andreas Damianou (ORCID: https://orcid.org/0000-0001-9383-5100)
- Zhu Liang (ORCID: https://orcid.org/0000-0001-8223-5003)
- Athina Grigoriou
- Vassilena Sharlandijeva
Institutions
- University of Oxford (GB)
- MRC Weatherall Institute of Molecular Medicine (GB)
- Alzheimer’s Research UK (GB)
- Oxford BioMedica (United Kingdom) (GB)
- Jenner Institute (GB)
Publication Details
- Journal
- STAR Protocols
- Published
- 2024-10-25
- DOI
- https://doi.org/10.1016/j.xpro.2024.103417
- Citations
- 2
- Primary Topic
- Biotin and Related Studies
- Type
- article
- Field-Weighted Citation Impact
- 0.65
Funders
- Pfizer
- Alzheimer’s Research UK
- China Scholarship Council
- Chinese Academy of Medical Sciences