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 chr...
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Veröffentlicht in: | STAR protocols 2024-12, Vol.5 (4), p.103417, Article 103417 |
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Sprache: | eng |
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Zusammenfassung: | 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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•Step-by-step APEX2-based proximity labeling protocol for capturing molecular snapshots•Procedure to probe protein complexes at different functional stages•Instructions for profiling the molecular environment during NLRP3 inflammasome assembly
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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. |
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ISSN: | 2666-1667 2666-1667 |
DOI: | 10.1016/j.xpro.2024.103417 |