PMID- 37578715
OWN - NLM
STAT- MEDLINE
DCOM- 20230815
LR  - 20240507
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Print)
IS  - 1064-3745 (Linking)
VI  - 2696
DP  - 2023
TI  - Methods to Measure NLR Oligomerization I: Size Exclusion Chromatography, 
      Co-immunoprecipitation, and Cross-Linking.
PG  - 55-71
LID - 10.1007/978-1-0716-3350-2_4 [doi]
AB  - Protein oligomerization is a common principle of regulating cellular responses. 
      Oligomerization of NLRs is essential for the formation of NLR signaling platforms 
      and can be detected by several biochemical techniques. 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 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. Native gel electrophoresis also allows detection of the NLR 
      oligomerization state by immunoblot. Chemical cross-linking covalently joins two 
      or more molecules, thus capturing the oligomeric state with high sensitivity and 
      stability. ASC oligomerization has been successfully used as readout for NLR/ALR 
      inflammasome activation in response to various PAMPs and 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.
CI  - (c) 2023. The Author(s), under exclusive license to Springer Science+Business 
      Media, LLC, part of Springer Nature.
FAU - Khare, Sonal
AU  - Khare S
AD  - Department of Academic Pathology, Department of Biomedical Sciences and Samuel 
      Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, 
      CA, USA.
FAU - Devi, Savita
AU  - Devi S
AD  - Department of Academic Pathology, Department of Biomedical Sciences and Samuel 
      Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, 
      CA, USA.
FAU - Radian, Alexander D
AU  - Radian AD
AD  - Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, 
      Northwestern University, Chicago, IL, USA.
FAU - Dorfleutner, Andrea
AU  - Dorfleutner A
AD  - Department of Academic Pathology, Department of Biomedical Sciences and Samuel 
      Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, 
      CA, USA.
FAU - Stehlik, Christian
AU  - Stehlik C
AD  - Department of Academic Pathology, Department of Biomedical Sciences and Samuel 
      Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, 
      CA, USA. christian.stehlik@csmc.edu.
LA  - eng
GR  - R01 AI134030/AI/NIAID NIH HHS/United States
GR  - R01 AI140702/AI/NIAID NIH HHS/United States
GR  - R01 GM071723/GM/NIGMS NIH HHS/United States
GR  - R01 AI165797/AI/NIAID NIH HHS/United States
GR  - R01 AI099009/AI/NIAID NIH HHS/United States
GR  - K01 AR066739/AR/NIAMS NIH HHS/United States
GR  - R01 AR064349/AR/NIAMS NIH HHS/United States
GR  - R01 AI120625/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Inflammasomes)
RN  - 0 (NLR Family, Pyrin Domain-Containing 3 Protein)
RN  - 0 (Lipopolysaccharides)
RN  - EC 3.4.22.- (Caspases)
RN  - EC 3.4.22.36 (Caspase 1)
RN  - 0 (Interleukin-1beta)
RN  - 0 (NLRP7 protein, human)
RN  - 0 (Adaptor Proteins, Signal Transducing)
SB  - IM
MH  - Mice
MH  - Animals
MH  - Humans
MH  - *Inflammasomes/metabolism
MH  - *NLR Family, Pyrin Domain-Containing 3 Protein/metabolism
MH  - Staphylococcus aureus/metabolism
MH  - Lipopolysaccharides
MH  - Chromatography, Gel
MH  - Immunoprecipitation
MH  - Caspases/metabolism
MH  - Caspase 1/metabolism
MH  - Interleukin-1beta/metabolism
MH  - Adaptor Proteins, Signal Transducing/metabolism
PMC - PMC11073631
MID - NIHMS1985323
OTO - NOTNLM
OT  - Blue native electrophoresis
OT  - Caspase-1 activity assay
OT  - Co-immunoprecipitation
OT  - Cross-linking
OT  - Inflammasome
OT  - NLR
OT  - Nod-like receptor
OT  - Oligomerization
OT  - Protein-protein interaction
OT  - Size exclusion chromatography
EDAT- 2023/08/14 12:43
MHDA- 2023/08/15 06:42
PMCR- 2024/05/06
CRDT- 2023/08/14 11:32
PHST- 2023/08/15 06:42 [medline]
PHST- 2023/08/14 12:43 [pubmed]
PHST- 2023/08/14 11:32 [entrez]
PHST- 2024/05/06 00:00 [pmc-release]
AID - 10.1007/978-1-0716-3350-2_4 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2023;2696:55-71. doi: 10.1007/978-1-0716-3350-2_4.