(A) Western blotting of CGNs treated with 50 pM TeNT alone (PC) or preincubated with the indicated TT104-Fab/TeNT molar ratio. After 12 hours, CGNs were lysed and immunoblotted for VAMP-2 and SNAP-25 as in Figure 2. Blots are representative of 3 independent experiments. (B) Survival curve for mice injected i.p. with either TeNT alone (4 ng/kg) or premixed with 5 times the molar excess of TT104-Fab. The number of mice in each group is indicated in the panel. (C) Scheme illustrating entry of the TeNT L chain into the neuronal cytosol via either canonical receptor–mediated cell uptake and translocation across the membrane of SVs triggered by the acidification of their lumen induced by proton pump activity of the V-ATPase (light blue), or low-pH translocation across the plasma membrane in the presence of bafilomycin A1 (Baf-A1). (D) Western blot analysis showing the inhibition of TeNT L chain membrane translocation by TT110-Fab. CGNs were incubated at 4°C for 15 minutes with either TeNT (10 nM) or TeNT preincubated with TT110-Fab. The culture medium was then replaced with a 37°C buffer for 10 minutes at pH 7.4 or pH 5.0. Samples were then incubated for 12 hours with normal medium (PC) or normal medium supplemented with Baf-A1 (100 nM). Membrane translocation was assessed according to VAMP-2 cleavage. SNAP-25 served as a loading control. Results are representative of 3 independent experiments. (E) ANS fluorescence binding experiment showing the pH-induced conformational change of TeNT blocked by TT110-Fab. TeNT (0.35 μM) or TeNT preincubated with TT110-Fab was incubated at pH 7.0 in the presence of 50 μM ANS and liposomes. The conformational change was triggered by lowering the pH with sequential addition of specific volumes of HCl and evaluated following the ANS fluorescence intensity at 470 nm. Results are representative of 2 independent experiments.