Bid has also been suggested to be cleaved by granzymes, calpains, and cathepsins in the loop connecting alpha helices 2 and 3, generating cleavage products that may also induce the release of caspase-activating factors from mitochondria [23] [29].
Calpain I cleaves purified mHDx-1 in vitro, generating cleavage products of the expected sizes, showing that this protease can act directly on HDx-1 and supporting the site prediction.
Cleavage of Bcl-2 and Bcl-x by caspases during the execution phase of apoptosis can generate cleavage products that facilitate apoptosis [ 27, 28].
Moreover, galactose treatment did not generate cleavage of caspase 3, the caspase active form, in contrast with cleavage observed with staurosporine, a classical apoptosis inducer and caspase 3 activator (Fig. 2B).
To avoid such size-related biases, we followed essentially the same protocol in generating cleavage products, but applied a modified Solexa paired-end DNA sequence library preparation protocol that does not include a size-selection step (Henikoff et al., 2011).
A similar strategy was used to generate cleavage-resistant forms of MKK3, MKK4, MKK6 and MKK7.
In a very similar in vitro cleavage assay reported previously, MMP-7 generated several cleavage products of E-cadherin with apparent molecular weights down to 80 kDa [29].
We show via in vitro assays that caspases are sufficient to cleave actin and tubulin, and our cleavage-specific antibodies recognize a neoepitope generated by cleavage.
