Biochemical and microscopic analysis of inflammasome complex formation.

Autor:	Swacha P; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden., Gekara NO; Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden. Electronic address: nelson.gekara@su.se., Erttmann SF; Department of Molecular Biology, Umeå University, Umeå, Sweden.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2019; Vol. 625, pp. 287-298. Date of Electronic Publication: 2019 Jun 10.
DOI:	10.1016/bs.mie.2019.05.014
Abstrakt:	Inflammasomes are multiprotein signaling platforms responsible for the maturation of pro-IL-1β and pro-IL-18 as well as the induction of an inflammatory cell death termed pyroptosis. Most inflammasomes consist of an upstream sensor, in most cases an adaptor protein (ASC) and inflammatory caspases such as caspase-1. Upon activation, sensor proteins oligomerize with adaptor proteins, forming large complexes called specks. These complexes can be stabilized and detected by Western blotting or fluorescence microscopy providing a direct evidence of inflammasome activation. Here we describe protocols for two complementary methods for detecting inflammasome complexes: (1) biochemical isolation and detection of ASC oligomers by Western blot analysis and (2) microscopic visualization of active caspase-1-ASC complexes. These protocols have successfully been applied in our recent study to unveil new regulatory mechanisms for different inflammasomes including the DNA sensor AIM2 (Erttmann et al., 2016). (© 2019 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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