Fig 2: VAMP-cleaving BoNT/DA inhibits AITC- and CAP-evoked CGRP releases from TGNs more extensively than BoNT/A, which proteolyses SNAP-25. Cultured TGNs were pre-treated with 100 nM BoNT/A, BoNT/DA, or control medium for 48 h, before determining spontaneous and evoked CGRP release over sequential 30 min periods. At the end of each experiment, a cohort of cells was lysed in SDS-PAGE sample buffer and subjected to Western blotting with a mixture of three different antibodies recognising syntaxin-1 (isoforms A and B), SNAP-25 (both intact plus cleaved forms), and VAMP (isoforms 1, 2, and 3), respectively (see Section 4 for details). (A) Representative Western blots show that BoNT/A cleaved SNAP-25, whereas BoNT/DA proteolysed and, thereby, diminished detectable VAMPs 1/2, and 3 (note that VAMPs 1 and 2 co-migrate). Syntaxin-1 is not affected by either of the toxins, so it was used as a loading control. (B) The fractions of SNAP-25 or VAMP1/2 cleaved were quantified by densitometric analysis (as described in Section 4); N = 5, n = 5. Note that the weak VAMP3 signals were not analysed. (C) The total content of CGRP (pg/well). (D) Histogram displaying spontaneous CGRP release, in control cells and those treated with the BoNTs, calculated as % of the total CGRP; N = 3, n = 16. (E) CGRP release evoked by various [AITC] for 30 min from TGNs pre-treated with BoNTs, expressed as % of requisite amounts elicited from non-intoxicated control cells; N = 2, n = 5. (F) After pre-treatment with the indicated BoNTs, TGNs were exposed to different [CAP], or (G) sequentially stimulated with 1 mM AITC (yellow) and then 1 µM CAP (red) for 30 min each. Evoked CGRP release is expressed as a % of control level (F) or in (G) % of the total content of this neuropeptide. Note, in (F) data for BoNT/A are from [41]. Data are presented as mean ± s.e.m. One- or two-way ANOVA was used in (D,G) followed by Bonferroni’s post hoc test; ## p < 0.01 for CAP-evoked in control vs. BoNTs; **** p < 0.0001 for AITC-evoked vs. BoNTs.