FIG 7 .

VHH-B8-LTIIa protects neurons from BoNT/A intoxication. (A) Schematic of VHH-B8-LTIIa. (B and C) A 1 nM concentration of BoNT/A (B) or BoNT/D (C) was incubated with rat cortical neurons at 37°C for 2 h, when toxin was removed and the indicated amount of VHH-B8-LTIIa (B8) was added to neurons for an additional 3 h. Cells were fixed and incubated with Alexa 647-wheat germ agglutinin (WGA; magenta) for 30 min as a cell marker. The IF assay stained for HA (red) and cleaved SNAP25 (SNAP25c, green) in BoNT/A-treated cells or intact VAMP2 (green) in BoNT/D-treated cells. (D) Cleavage was quantified by measuring the SNAP25c/WGA ratio of fluorescence intensities for BoNT/A-treated cells and the VAMP2/WGA ratio of fluorescence intensities for BoNT/D-treated cells. (E) BoNT/A or BoNT/D (1 nM) was incubated with rat cortical neurons at 37°C for 2 h, when toxin was removed and the indicated amount of VHH-B8-LTIIa (B8) was added with neurons overnight. Cleavage was quantified as described for panel D. Data were analyzed as SEM by two-tailed Student’s t test. *, P < 0.05; **, P < 0.005. Bar, 20 µm. NS, not significant; o/n, overnight.