Analysis of CYLD Proteolysis by CASPASE 8 in Bone Marrow-Derived Macrophages
Previous studies have demonstrated that CASPASE 8 can generate a prosurvival signal by inhibiting necroptosis via the cleavage of the deubiquitinating enzyme CYLD. Cleavage of CYLD at D215 results in the generation of a 25 kD N-terminal fragment and degradation of the C-terminal fragment containing the catalytic domain. Since CYLD is required for TNF-induced necroptosis, its proteolysis is necessary and sufficient to suppress necroptosis and generate a survival signal. Here we describe how to visualize CYLD proteolysis by western blot analysis, as a measure of CASPASE 8 activity and inhibition of necroptosis.
Key wordsBone marrow-derived macrophages Necroptosis CYLD Proteolysis Cleavage
This work was supported by grants AI052417 and DK072201 from the NIH, and a Senior Research Award from the Crohn’s and Colitis Foundation of America (CCFA).
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