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. 2015 Jul 24;10(7):e0133737. doi: 10.1371/journal.pone.0133737

Fig 3. Inhibition of BoNT/A entry into human neurons by endocytosis inhibitors specific for clathrin dependent endocytosis.

Fig 3

(a) Human iPSC derived neurons (iCell Neurons) were treated with the indicated endocytosis inhibitors for 30 min at 37°C. The cells were then exposed to 200 U of BoNT/A1 / well for 7 h, and cell lysates analyzed for SNAP-25 cleavage by Western blot. B: Human iPSC derived neurons (iCell Neurons) were exposed to 1000 U / well of BoNT/B to achieve full VAMP2 cleavage (top panel). Toxin was removed, and cells were treated with the indicated endocytosis inhibitors for 30 min at 37°C. The cells were then exposed to 200 U of BoNT/A1 / well for 5 min in either culture media or cell stimulation media, followed by toxin removal and further incubation for 8 h. Cell lysates were analyzed for SNAP-25 cleavage by Western blot. SNAP-25 cleavage in depolarized (cell stimulation media) or not depolarized (culture media) cells is shown in the Western blot, and the respective inhibitors are indicated in the table.