Figure 4.

Fusion of early endosomes from PC12 cells is not inhibited by preincubation of the cell-free extract with TeNT-L chain and BoNT/C1-L chain. (A) Aliquots of the toxin-treated extracts were analyzed by SDS-PAGE and immunoblotting for substrate breakdown. In the first lane untreated cell-free extract was loaded. Note that the syntaxin signal is variable; however, partial breakdown is evident by the appearance of bands of increased mobility reflecting cleavage products. No higher degree of breakdown was observed when the BoNT/C1 L chain concentration was increased 50-fold (our unpublished results). (B) Cell-free extracts were incubated for 30 min before the fusion assay with 10 nM TeNT and BoNT/C1, respectively, using either active (act.) or heat-inactivated (inact.) recombinant toxin L chains. The data were derived from five independent experiments.