Abstract
Oligomerization of nod-like receptors (NLRs) can be detected by several biochemical techniques dependent on the stringency of protein–protein interactions. Some of these biochemical methods can be combined with functional assays, such as caspase-1 activity assay. Size exclusion chromatography (SEC) allows separation of native protein lysates into different sized complexes by fast protein liquid chromatography (FPLC) for follow-up analysis. Using co-immunoprecipitation (co-IP), combined with SEC or on its own, enables subsequent antibody-based purification of NLR complexes and associated proteins, which can then be analyzed by immunoblot and/or subjected to functional caspase-1 activity assay. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. Apoptosis-associated speck-like protein containing a caspase activation domain (ASC) oligomerization has been successfully used as readout for NLR or AIM2-like receptor (ALR) inflammasome activation in response to various pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) in human and mouse macrophages and THP-1 cells. Here, we provide a detailed description of the methods used for NLRP7 oligomerization in response to infection with Staphylococcus aureus (S. aureus) in primary human macrophages, co-immunoprecipitation and immunoblot analysis of NLRP7 and NLRP3 inflammasome complexes, as well as caspase-1 activity assays. Also, ASC oligomerization is shown in response to dsDNA, LPS/ATP, and LPS/nigericin in mouse bone marrow-derived macrophages (BMDMs) and/or THP-1 cells or human primary macrophages.
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Acknowledgments
This work was supported by the National Institutes of Health (GM071723, AI099009, AI120618 and AR064349 to C.S., AI120625 to C.S. and A.D. and AR066739 to A.D.) and the American Heart Association (12GRNT12080035 to C.S.).
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Khare, S., Radian, A.D., Dorfleutner, A., Stehlik, C. (2016). Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking. In: Di Virgilio, F., Pelegrín, P. (eds) NLR Proteins. Methods in Molecular Biology, vol 1417. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3566-6_8
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DOI: https://doi.org/10.1007/978-1-4939-3566-6_8
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