Reagents/supplies

Reagents included ammonium bicarbonate (Acros Organics, catalog no. 393212500), d-desthiobiotin (MilliporeSigma, catalog no. D1411), dl-dithiothreitol (DTT) (MilliporeSigma, catalog no. D0632-5G), formaldehyde, 16% solution (Electron Microscopy Sciences, catalog no. 15710), formic acid (Thermo Fisher Scientific, catalog no. A117-50), l-glutathione oxidized (MilliporeSigma, catalog no. G4376), l-glutathione reduced (MilliporeSigma, catalog no. G4251), imidazole (Thermo Fisher Scientific, catalog no. BP305-50), β-mercaptoethanol (Bio-Rad, catalog no. 1610710), polyethylenimine (MilliporeSigma, catalog no. 408727), polyethylenimine/laminin-coated glass coverslips (MilliporeSigma), phosphate-buffered saline (PBS)–G (MilliporeSigma, catalog no. G0411), 10% Triton X-100 (Surfact-Amps X-100, Thermo Fisher Scientific, catalog no. 28314), 1× Dulbecco’s PBS (Thermo Fisher Scientific, catalog no. 21-030-CV), 2% sterile gelatin solution (MilliporeSigma, catalog no. G1393), Criterion XT 4–12% Bis-Tris gel (Bio-Rad, catalog no. 3450124), XT MES Running Buffer (Bio-Rad, catalog no. 1610789), 4× Laemmli sample buffer (Bio-Rad, catalog no. 1610747), Coomassie Blue R-250 stain solution (Teknova, catalog no. C1050), 4–12% Bis-Tris NuPAGE gel (Thermo Fisher Scientific, catalog no. NP0323BOX), 20× NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, catalog no. NP000102), 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific, catalog no. NP0007), BCA assay kit (Thermo Fisher Scientific, catalog no. 23227), nitrocellulose membranes (Bio-Rad, catalog no. 162–0168), thick blot paper (Bio-Rad, catalog no. 1703956), normal goat serum (Thermo Fisher Scientific, catalog no. 16210–072), SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, catalog no. 34080), BL21 (DE3) CodonPlus-RIL cells (Agilent Technologies, catalog no. 230245), pRK793 6-His-TEV protease plasmid (Addgene, catalog no. 8827), saponin (MilliporeSigm, catalog no. 558255), Neurobasal Medium (Thermo Fisher Scientific, catalog no. 21103049), B27 Supplement (Thermo Fisher Scientific, catalog no. 17504044), NbActiv4 medium (BrainBits LLC, catalog no. Nb4-500), E18 rat cortical tissue (BrainBits LLC, catalog no. SDECX), SF-900 II serum-free medium (Thermo Fisher Scientific, catalog no. 10902088), NeuroCult SM1 Supplement (STEMCELL Technologies, catalog no. 05711), anti–neurofilament-M (NFIL; MilliporeSigma, catalog no. MA1621), IgG-free bovine serum albumin (BSA) (MilliporeSigma, catalog no. A0336), glass coverslips coated with poly-d-lysine (MilliporeSigma, catalog no. P6407), anti–β-actin mouse IgG2a (MilliporeSigma, catalog no. A5316), anti–EEA-1 mouse IgG (BD Biosciences, catalog no. 610457), anti–hemagglutinin (HA) 3F10 rat IgG (MilliporeSigma, catalog no. 11867423001), anti–lysosomal-associated membrane protein–1 (LAMP-1) rabbit IgG (MilliporeSigma, catalog no. AB2971), anti–Rab-7 mouse monoclonal antibody (mAb) (Santa Cruz Biotechnology, catalog no. sc-376362), anti–mitogen-activated protein 2 (MAP2) chicken IgY (Abcam, catalog no. ab5392), anti–SNAP-25 mouse IgG (Synaptic Systems, catalog no.111011), anti-STX1 mouse IgG (Synaptic Systems, catalog no. 106011), anti-rat Tau mouse IgG2b (BD Biosciences, catalog no. 610672), Alexa Fluor 555 goat anti-human IgG (Thermo Fisher Scientific, catalog no. A-21433), Alexa Fluor 555 goat anti-mouse IgG2b (Thermo Fisher Scientific, catalog no. A-21422), Alexa Fluor 647 goat anti-mouse IgG2b (Thermo Fisher Scientific, catalog no. A-21242), Alexa Fluor 647 goat anti-rabbit IgG (Thermo Fisher Scientific, catalog no. A-21245), Alexa Fluor 488 goat anti-human IgG (Thermo Fisher Scientific, catalog no. A-11013), horseradish peroxidase (HRP) anti–E-tag mAb (GE Healthcare), 3,3′,5,5′-tetramethylbenzidine (MilliporeSigma, catalog no. 860336), anti-OLLAS tag (rat mAb; Thermo Fisher Scientific, catalog no. MA5-16125), c-myc tag (mouse mAb; Thermo Fisher Scientific, catalog no. 13-2500), E-tag [rabbit polyclonal antibody (pAb); Abcam, catalog no. ab3397], anti-BoNT/C1 LC [human mAb 4C10.2; J. Marks, University of California San Francisco (UCSF)], anti-BoNT/C1 HC (human mAb 8DC1.2; J. Marks, UCSF), anti-HA tag (mouse mAb 3F10; MilliporeSigma, catalog no. 11867423001), rabbit anti-BoNT/A1 (pAb Pol001; Statens Serum Institut, Denmark), BoTest A/E BoNT Detection Kit (BioSentinel Inc., catalog no. A1004), sheep pAb against BoNT/C1 receptor-binding domain (R&D Systems, catalog no. AF5425), ProLong Gold 4′,6-diamidino-2-phenylindole (DAPI) mounting media (Thermo Fisher Scientific, catalog no. {"type":"entrez-protein","attrs":{"text":"P36935","term_id":"549826","term_text":"P36935"}}P36935), Ni²⁺-NTA Sepharose fast flow (IBA GmbH, catalog no. 2-3206-025), StrepTactin Sepharose fast flow (IBA GmbH, catalog no. 2-1208-025), HiLoad 26/600 Superdex 200 pg gel filtration column (GE Healthcare Life Sciences, catalog no. 28989336), and 0.22-μm filters (MilliporeSigma, catalog no. UFC30GV0S). All other chemicals were purchased from either Thermo Fisher Scientific or MilliporeSigma, unless indicated otherwise.