Fig. 5. Sbr is not required for Ca²⁺ to trigger changes in synaptotagmin I, SNAP-25 and syntaxin. (A) Membranes, prepared from intact control and BoNT/B-treated chromaffin cells, were subjected to SDS-PAGE followed by western blotting. (B-F) Toxin-free () and BoNT/B-treated ([UNK]) chromaffin cells were permeabilised by exposure to digitonin in KGEP, with the inclusion of incremental free [Ca²⁺]. Following a 15 minute interval, aliquots were removed and assayed (±s.d.; n=4) for catecholamine (B); then 100 µg/ml trypsin was added and 30 minutes later 2 mM PMSF was included and a membrane fraction prepared. The samples were boiled for 2 minutes prior to SDS-PAGE and western blotting (C); the amounts of synaptotagmin (D), syntaxin (E) and SNAP-25 (F) present in each were quantified and normalised as a percentage of the largest value in each [Ca²⁺] series. Plotted data are representative of results obtained on at least three separate occasions.