Fig. 5.

Bafilomycin, methylamine hydrochloride and ammonium chloride inhibit DNA transfection reagent-mediated enhancement of BoNT/A holotoxin and Lc internalization into cells. Primary RCGNs, neuronal cell lines N2A, M17 and non-neuronal cell lines 293HEK, HIT-T15 were treated with bafilomycin for 2 hrs and washed with DPBS. Cells were then exposed to 10 nM of BoNT/A toxin for 4 or 24 hrs or 30 nM of Lc438 for 24 hrs, + or − pre-incubation with the FuGene-HD transfection reagent. Control cells were incubated with 10 nM of BoNT/A toxin or 30 nM of Lc438, + or − pre-incubation with FuGene-HD, without prior exposure of the cells to bafilomycin. Cell extracts were prepared and the extent of BoNT/A intoxication was assessed by Western blotting to monitor SNAP25 cleavage. B. N2A, M17 and HIT-T15 cells were treated +/− bafilomycin and then transfected 24 hrs with pcDNA/CFP in FuGene-HD as above. Cell extracts were resolved by SDS-PAGE and CFP was detected by Western blotting. C. M17 cells were treated with 10 mM methylamine hydrochloride for 1 h or 8 mM ammonium chloride (NH₄Cl) for 2 hrs and then exposed to 10 nM Lc438 with (+) or without (−) FuGene-HD transfection reagent (1:3) for 4 hrs. Cell extracts were prepared and the extent of BoNT/A intoxication was assessed by Western blotting to monitor SNAP25 cleavage.