Protocol for reconstitution of oligomeric assembly of NAIP5-NLRC4 inflammasome in vitro

Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023).(1)