Fig. 4: Mechanistic assay using the CB-assay.

a Increasing cell uptake by GT1b addition. rBoNT/A (1 pM) was incubated 24 h in the absence (green triangles) or presence of GT1b (blue diamonds) and the action of the toxin was determined by measuring the percentage of BoNT sensor4 cleavage. Data points are plotted as individual values for each independent experiment and mean (4 experiments performed in triplicates). Controls are cells with no treatment corresponding to basal conditions (black circles) and cells incubate only with GT1b (pink squares). b Blocking of toxin action by ConA. rBoNT/A (1 nM) was incubated 10 min in stimulating conditions (60 nM KCl). After toxin washout and 4 h in the absence (green triangles) or presence (blue diamonds) of ConA, the percentage of BoNT sensor4 cleavage was determined. Data points are plotted as individual values for each independent experiment and mean (4 experiments performed in triplicates). Controls are cells with no treatment corresponding to basal conditions (black circles) and cells incubate only with ConA (pink squares). Data points are mean of n = 4 independent experiments in triplicate. Statistical ordinary one-way ANOVA multiple comparisons: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, P > 0.05 not significant (NS). Error bars show SEM.