Figure 5

EGA does not inhibit the translocation and the reduction step.

(a) PC12 cells expressing the lumenal domain of synaptotagmin I on their surface were pre-incubated with a mixture of gangliosides for 24 hrs. Cells were washed and, where indicated, treated with 12.5 μM EGA or vehicle for 30 min at 37 °C. Thereafter, BoNT/B (10 nM) was added in the cold for 15 min. Cells were then washed and incubated with medium A buffered at indicated pH at 37 °C for 10 min, in the presence or absence of EGA. Then, cells were washed and the incubation in culture medium containing 50 nM Bafilomycin A1 and the same concentration of EGA prolonged for 24 hrs at 37° C. The translocation of BoNT/B was assessed by monitoring the cleavage of VAMP2, determined via western blotting (top panel) and quantified (bottom panel) through the densitometry of VAMP2 as a ratio to SNAP25 staining which served as internal control, taking the value in non-treated cells (NC) as 100%. All data are presented as mean values and error bars indicated the standard deviation obtained from three independent experiments (ns – non significant). (b) CGNs were treated with 12.5 μM EGA or vehicle for 30 min at 37 °C. Then, neurons were incubated with BoNT/D (2.5 pM) at 4 °C for 15 min, washed and incubated at 37 °C with buffers at different pH value (7.4 or 4.5) for 10 min; after washing, the neurons were incubated for 24 h with standard medium in the presence of 50 nM bafilomycin A1 and where indicated EGA. Then the SNARE protein content was estimated by immunoblotting with specific antibodies. Values are reported as the ratio between the staining with the antibody specific for VAMP2 and the staining with an antibody specific for SNAP25 and normalized vs. non-treated neurons (NC). All data are presented as mean values and error bars indicated the standard deviation obtained from three independent experiments (ns – non significant).