Figure 2.

EGA prevents the TeNT-induced cleavage of VAMP-2 in cultured neurons. CGNs were incubated with different concentrations of EGA at 37 °C for 30 min. TeNT 2 nM was added for 10 min, cells were washed, and culture medium with the same concentration of inhibitor was restored. Incubation was then prolonged for 4 h at 37 °C. (A) Representative immunoblot showing the relative amount of VAMP-2, SNAP-25 and Syntaxin-1A in CGNs in control conditions (vehicle), upon treatment with TeNT alone (PC) and upon pre-treatment with decreasing amounts of EGA before TeNT addition. (B) Quantification of VAMP-2 signal in Western blots reported as a percent vs. vehicle and calculated as a ratio of STX-1A staining used as loading. SD values derive from three independent experiments. Graphs show mean ± SD. Significance was calculated by a two tailed Student’s t-test compared to neurons treated with TeNT alone (*** p < 0.001; * p < 0.05) (C) Representative immunofluorescence for VAMP-2 staining in CGNs treated as in (A). EGA was used at 12.5 µm. The image is representative of three independent experiments. Scale bar is 20 µm.