Figure 2.

Determination of the cleavage site of vMLC1 and its specificity for caspase-3. (A) Cleavage of purified human vMLC1 (10 μg) by recombinant active caspase-3 (20 ng/μl, 1 hr, 37°C), analyzed by SDS/16.5% PAGE and Coomassie blue staining. (B) Immunoblot analysis of wild-type vMLC1, vMLC1^mD132A, and vMLC1^mE135S proteins expressed in COS-7 cells, after incubation of the cell extracts with recombinant active caspase-3 (20 ng/μl, 1 hr, 37°C). The first line (c) shows extracts from COS-7 cells transfected with control vector plasmid. Mouse monoclonal antibody directed against the residues 1 to 8 (clone F109.17A5) was used for immunodetection. (C) A diagram showing the caspase-3 cleavage site at the C-terminal side of E¹³⁵ of human, rabbit, rat, and chicken vMLC1. (D) Immunoblot analysis of vMLC1 cleavage in protein extracts from rabbit left ventricle, incubated with different human recombinant active caspases (25 ng/μl) for 1 hr.