Fig. 3. The Ca²⁺ dependencies of exoytosis and for the acquisition by SNAREs of trypsin resistance are unaffected following SNAP-25 truncation by BoNT/A. Chromaffin cells, in the absence () or presence ([UNK]) of 66 nM BoNT/A (see Materials and Methods), were permeabilised and exposed for 15 minutes to a range of free [Ca²⁺] (MgATP was not included), then catecholamine release was assayed as described in Fig. 1; the results are plotted in (A). Cells were incubated without (B) or with (C) trypsin (100 µg/ml final concentration) for a further 30 minutes, and 2 mM PMSF added before a membrane fraction was prepared. The latter samples were boiled for 10 minutes, subjected to SDS-PAGE and analysed by western blotting under identical conditions. The amount of SNAREs remaining in the trypsin-treated cells was quantified (see Materials and Methods) using photographic exposures optimised to show the Ca²⁺ dependence of trypsin resistance (i.e. using photographs in which the signals from BoNT/A-treated cells had been developed for longer than shown in C). The signal intensities were quantified as in Fig. 1 for control (open symbols) and BoNT/A-poisoned cells (closed symbols) and plotted for synaptotagmin I (D), syntaxin (E), SNAP-25 (F) and synaptobrevin (G). Plotted data are representative of results obtained on at least three separate occasions.