Abstract
Gasdermin D (GSDMD) is a recently identified pore-forming protein that is crucial for the execution of pyroptosis, a highly inflammatory form of cell death. GSDMD contains an N-terminal and a C-terminal domain that are separated by a proteolysis-sensitive linker. Upon cleavage of this linker by inflammasome-activated caspases, the N-terminal domain of GSDMD oligomerizes and forms pores at the plasma membrane, allowing cell swelling and subsequently membrane rupture to mediate pyroptosis. GSDMD is a key substrate of inflammatory caspases downstream of inflammasome activation and is driving various pathologies. Here, we describe a simple method to study GSDMD cleavage following canonical inflammasome activation in murine primary macrophages and neutrophils and human cell lines using immunoblotting.