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.
