In vitro gasdermin D cleavage assay

To examine cleavage of gasdermin D, about 7.5 μM purified murine gasdermin D was incubated with 2.5 μM purified caspase-11 (p22/p10 form) (33) at 37°C for 30 min in buffer A [20 mM tris-HCl (pH 8.0) and 150 mM NaCl] with or without 2 or 4 μg of purified Stx2. The cleavage was stopped by adding SDS loading buffer to the reaction mixture, followed by boiling at 95°C for 5 min. The samples were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining.