Fig. 2. SNAREs in cells subjected to run-down do not acquire resistance to trypsin. (A) Cells were permeabilised with digitonin in KGEP containing Ca²⁺ at the indicated concentrations () or with digitonin in KGEP lacking Ca²⁺ and maintained for 30 minutes before the addition of the cation ([UNK]). In both cases, aliquots were removed for catecholamine assay 15 minutes after the application of Ca²⁺ and released catecholamine was calculated (±s.d., n=4; some error bars are obscured by symbols). Trypsin was immediately added (100 µg/ml final concentration) and incubated for 30 minutes before adding 2 mM PMSF, harvesting the cells and isolating a membrane-enriched fraction. The latter samples were boiled for 2 minutes before being subjected to SDS-PAGE and immunoblotting (B), as described in Fig. 1. In (C), cells were permeabilised and exposed for 15 minutes to incremental [Ca²⁺] before the addition of 10 µg/ml proteinase K. After a further 30 minutes, 2 mM PMSF was added, membranes were prepared and analysed by western blotting, as above.