Fig. 3. BoNT/A alters the ratio of SNAREs in complexes. (A) Control and BoNT/A-pre-treated chromaffin cells (see Materials and Methods) were permeabilised with 20 µM digitonin in KGEP, in the absence or presence of 20 µM Ca²⁺. After 45 minutes, 2 mM PMSF was added, the cells were lysed and a membrane-enriched fraction prepared. Two-dimensional SDS-PAGE and western blotting was performed, as described in Figs 1 and 2. (B) Cells were treated as in (A), except that 15 minutes after permeabilisation the cells were exposed to 100 µg/ml trypsin for 30 minutes.