A Streamlined Method for Detecting Inflammasome-Induced ASC Oligomerization Using Chemical Crosslinking

Methods Mol Biol. 2023:2691:155-164. doi: 10.1007/978-1-0716-3331-1_12.

Abstract

The apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) functions as the integral adaptor protein between inflammasome sensors such as NOD-like receptor protein 3 (NLRP3) and the inflammatory caspase, caspase-1. Inflammasome sensor triggering allows recruitment of ASC and the formation of long amyloid-like ASC oligomers that enable binding and proximity-induced activation of caspase-1. The detection of ASC oligomerization thus constitutes a highly specific and direct test for inflammasome complex formation and activation. Here, we describe a simplified and streamlined method for the detection of ASC oligomers via Western blotting, using the chemical crosslinking reagent disuccinimidyl suberate.

Keywords: ASC; Caspase-1; Cell death; Inflammasome; Inflammation; Innate immunity; Interleukin-1; NLRP3; Pyroptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • CARD Signaling Adaptor Proteins / genetics
  • CARD Signaling Adaptor Proteins / metabolism
  • Caspase 1 / metabolism
  • Caspases / metabolism
  • Inflammasomes* / metabolism
  • Interleukin-1beta / metabolism
  • NLR Family, Pyrin Domain-Containing 3 Protein* / metabolism

Substances

  • Inflammasomes
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Caspase 1
  • Caspases
  • CARD Signaling Adaptor Proteins
  • Interleukin-1beta