title: Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking.
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.
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 60611, USA.
authors.forename: Sonal
authors.initials: S
authors.lastname: Khare
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 60611, USA.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Radian
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 60611, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Dorfleutner
authors.affiliation: Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 60611, USA. c-stehlik@northwestern.edu.
authors.forename: Christian
authors.initials: C
authors.lastname: Stehlik
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000818:Animals; D053468:CARD Signaling Adaptor Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D006801:Humans; D047468:Immunoprecipitation; D008264:Macrophages / Q000166:cytology; D051379:Mice; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000737:chemistry*; D055503:Protein Multimerization; D013203:Staphylococcal Infections / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000502:physiology; D000074084:THP-1 Cells
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D053468:CARD Signaling Adaptor Proteins; D003432:Cross-Linking Reagents; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C502448:NLRP7 protein, human; C402202:PYCARD protein, human
keywords: Caspase-1 activity assay; Co-immunoprecipitation; Cross-linking; Inflammasome; NLR; Nod-like receptor; Oligomerization; Protein–protein interaction; Size exclusion chromatography