Fig. 3. Giantin and syntaxin 5 are cleaved by caspase-3. (A) Cytosols from HeLa cells were pre-incubated in the absence (control) or presence (apoptotic) of 10 µM cytochrome c for 90 minutes at 37°C to activate endogenous caspases. Golgi membranes were incubated with these control or apoptotic cytosols in the absence or presence of 80 µg/ml Casputin^TM for 4 hours at 37°C and analysed by SDS-PAGE and immunoblotting. Full-length (FL) and caspase cleavage products (^*) are marked. Filled circles indicate minor giantin cleavage products. (B) In vitro translated ³⁵S-labelled control proteins, giantin or syntaxin 5 were incubated with purified recombinant caspases at the indicated concentrations for 2 hours at 30°C prior to SDS-PAGE and autoradiography. Defined caspase cleavage products (^*) are marked. Additional cleavage products that may be non-specific (filled circles) are also marked. The open triangle indicates an additional giantin cleavage product seen only with caspase-2 that may be the cleavage partner for P3 (see also Fig. 5C). Control proteins were PARP (for caspase-3 and -7 at 0, 0.025, 0.1, 0.25, 0.5, 1, 2 and 4 nM) and pro-caspase-2 (for caspase-2 at 0, 0.5, 1, 2.5, 10, 25, 100, 250 nM).