Over the past five years it has become clear that protease activation is the single most important event that characterizes the process of apoptosis. In the present chapter, we briefly review the evidence that proteases—particularly caspases—play an important role in apoptosis. We then discuss various methods that have been applied to the study of caspases and their precursors, including immunoblotting, activity assays, and affinity labelling. We also describe various polypeptides whose cleavage can be used as a marker for caspase activation in situ. Finally, we outline an approach for determining whether a particular cleavage is mediated by caspases, focusing on the potential pitfalls in relying solely upon in vitro cleavage assays or inhibitor studies.
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