Inflammatory Caspases: Activation and Cleavage of Gasdermin-D In Vitro and During Pyroptosis

Gasdermin-D (also known as GSDMD), the newly identified executioner of pyroptotic cell death, is cleaved by activated Caspase-1 downstream of canonical inflammasome activation or caspase-4, 5, and 11 upon their ligation and activation by cytosolic LPS. Upon a single cleavage between the two domains in Gasdermin-D, the N-terminal domain binds to membrane lipids and lyses cells by forming pores of an inner diameter of 10-14 nm within the membrane. The inter-domain cleavage of Gasdermin-D is a reliable marker for the activation of inflammatory caspases and cell Pyroptosis. Here, we describe the methods for examining Gasdermin-D cleavage by activated inflammatory caspases in vitro and upon inflammasome activation in vivo.