(B) An in vitro cleavage assay was performed through mixing GSDMD and its mutants with caspase-1 p20/p10 complex, followed by immunoblotting with antibodies against the indicated protein tags. β-actin served as a loading control. FLAG-tagged GSDMD and its mutants were immunoprecipitated from HEK293T cells using anti-FLAG coupled magnetic beads. HA-tagged p20 and p10 were immunoprecipitated from HEK293T cells using anti-HA coupled agarose beads. Data information: Experiments were repeated for three times with similar results. 