PMID- 27221486
OWN - NLM
STAT- MEDLINE
DCOM- 20171213
LR  - 20181113
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Print)
IS  - 1064-3745 (Linking)
VI  - 1417
DP  - 2016
TI  - Measuring NLR Oligomerization I: Size Exclusion Chromatography, 
      Co-immunoprecipitation, and Cross-Linking.
PG  - 131-43
LID - 10.1007/978-1-4939-3566-6_8 [doi]
AB  - 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.
FAU - Khare, Sonal
AU  - Khare S
AD  - Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, 
      Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 
      60611, USA.
FAU - Radian, Alexander D
AU  - Radian AD
AD  - Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, 
      Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 
      60611, USA.
FAU - Dorfleutner, Andrea
AU  - Dorfleutner A
AD  - Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, 
      Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 
      60611, USA.
FAU - Stehlik, Christian
AU  - Stehlik C
AD  - 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.
LA  - eng
GR  - R01 GM071723/GM/NIGMS NIH HHS/United States
GR  - R21 AI120618/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 (Adaptor Proteins, Signal Transducing)
RN  - 0 (CARD Signaling Adaptor Proteins)
RN  - 0 (Cross-Linking Reagents)
RN  - 0 (NLR Family, Pyrin Domain-Containing 3 Protein)
RN  - 0 (NLRP7 protein, human)
RN  - 0 (PYCARD protein, human)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing/*chemistry
MH  - Animals
MH  - CARD Signaling Adaptor Proteins/*chemistry
MH  - Chromatography, Gel
MH  - Chromatography, Liquid
MH  - Cross-Linking Reagents
MH  - Humans
MH  - Immunoprecipitation
MH  - Macrophages/cytology
MH  - Mice
MH  - NLR Family, Pyrin Domain-Containing 3 Protein/*chemistry
MH  - Protein Multimerization
MH  - Staphylococcal Infections/*metabolism
MH  - Staphylococcus aureus/physiology
MH  - THP-1 Cells
PMC - PMC5058782
MID - NIHMS819945
OTO - NOTNLM
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- 2016/05/26 06:00
MHDA- 2017/12/14 06:00
PMCR- 2017/01/01
CRDT- 2016/05/26 06:00
PHST- 2016/05/26 06:00 [entrez]
PHST- 2016/05/26 06:00 [pubmed]
PHST- 2017/12/14 06:00 [medline]
PHST- 2017/01/01 00:00 [pmc-release]
AID - 10.1007/978-1-4939-3566-6_8 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2016;1417:131-43. doi: 10.1007/978-1-4939-3566-6_8.