Citations

Reagents:

… Botulinum toxins A and B were purchased from List Biologicals Laboratories, Campbell, CA. …

Study of the paralytic effect of BoNTs on detrusor contractions:

After intensive washings and after at least 20 min of stable contractions, BoNTs were added to the baths, with final concentrations of 0.1 to 5 nmol/L. Each strip was exposed to only one concentration of BoNT. Amplitude of contractions was measured until 90% of the signal was abolished, or for 4 h if this value was not reached (for low concentrations). …

Lung culture, drug treatments and imaging for morphometry:

… Lung cultures were treated with the following additions to the media to achieve final concentrations as follows: …botulinum toxin, serotype A (BTx): 50, 100, or 200 ng/mL (List Biologicals #128A, Campbell, California, USA). BTx was stored at 4°C (in a locked refrigerator), reconstituted at 100 ng/µL in water with 1 mg/mL BSA and handled in a fume hood. …

3D-EM of the L-PTC and the HA complex

The L-PTC of BoNT/A was obtained from List Biological Laboratories, Inc. (Campbell, California) and Miprolab GmbH (Gttingen, Germany). …For the L-PTC, no symmetry was imposed throughout the 3D reconstruction and refinement, while for the HA complex, a C3 symmetry was imposed. Refinement was terminated when no significant changes could be visually detected. A data set of 15,140 particles was used for the final reconstructed map of the L-PTC, for which the resolution was estimated to be 30.8 based on the resolution criteria of Fourier shell correlation (FSC) at 0.5 cutoff. …

Inhibition assays were performed for the recombinant light chain of BoNT/A, which is catalytically active, while binding assays used non-activated BoNT/A heavy and light chain complex. Confirmation of binding to whole BoNT/A was important because the catalytic site is expected to be partially occluded in the whole BoNT/A, which may affect efficacy of the inhibitor in vivo. …

…Toxin was obtained from List Biological Laboratories