Journal: iScience

Article Title: Neuronal SNAP-23 is critical for synaptic plasticity and spatial memory independently of NMDA receptor regulation

doi: 10.1016/j.isci.2023.106664

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-SNAP-23 , Novus Biologicals , Cat #. NBP2-67157; RRID: AB_2809999.

Techniques: Recombinant, Software, Microscopy

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A ) Illustration of the Caco-2 2D cell monolayer transwell system. Caco-2 cells were seeded into transwell inserts (3 µm pore size) and cultivated until the TEER had reached > 300 Ω•cm². Subsequently, the monolayer was infected with the Y. pseudotuberculosis wildtype strain YPIII at an MOI of 50. Differentiation (e.g., microvilli formation; left panel below), monolayer formation with adherent bacteria (right panel below), and barrier integrity (both middle panels) were visualized by electron microscopy. Created in BioRender. Dersch, P. (2026). ( B,C ) Confocal laser scanning microscopy (C-LSM) of Caco-2 monolayer infected with Y. pseudotuberculosis strain YPIII pFU92 expressing GFP (green) and stained with DAPI (nuclei-blue), and CellMask Plasma Membrane Red (CMPR) (red), and antibodies against glycoprotein-2 (GP-2) (yellow). Orthogonal XZ and YZ planes ( B ) and the apical surface of the infected Caco-2 monolayer ( C ) are shown. ( D ) Illustration of the methods used to determine the adherent/cell-associated number and intracellular number of Y. pseudotuberculosis in the Caco-2 cell monolayer after infection. The monolayer was infected with 2.5 x 10 6 bacteria of Y. pseudotuberculosis wildtype strain YPIII (wt) or the virulence plasmid-negative strain YP12 (ΔpYV) (MOI of 50), incubated for 3 h, and (i) washed with PBS and lysed to determine the number of cell-associated bacteria post-infection (pi), or (ii) gentamicin was added for an additional 2-7 h (i.e., 5 h and 10 h post-infection) before the cells were lysed, and the number of cell-associated or intracellular bacteria was quantified by plating serial dilutions on LB plates. Created in BioRender. Dersch, P. (2026). ( E ) The CFUs of the cell-associated and intracellular bacteria at the indicated time points are given. ( F ) Illustration of the methods used to determine the number of Y. pseudotuberculosis that egressed on the basolateral side of the Caco-2 cell monolayer. ( G ) 7 h after gentamicin was added to the infected monolayer (i.e., 10 h post-infection), the medium containing the egressed bacteria was collected and plated on LB plates. Created in BioRender. Dersch, P. (2026). ( E,G ) The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Pore Size, Infection, Bacteria, Electron Microscopy, Confocal Laser Scanning Microscopy, Expressing, Staining, Clinical Proteomics, Membrane, Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A ) Caco-2 2D monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt) (upper panel) or the YP147 (Δ cnfY ) mutant strain (lower panel). After 2, 3, or 4 h post-infection (p.i.), gentamicin was added to kill extracellular bacteria. Every 30 minutes post-infection, the medium from the basolateral chamber was collected over 10 hours and replaced. The removed medium was then plated on LB plates to determine the CFUs of egressed bacteria at the indicated time point. The mean +/- SEM of three independent experiments is shown. ( B-E ) Caco-2 2D monolayer was infected with Y. pseudotuberculosis wildtype strain YPIII, and monolayer samples were taken after 3.5 h and analyzed by transmission electron microscopy. Cell-adherent bacteria ( B ), as well as intracellular Yersiniae ( C ) were identified, indicated by black arrows. ( D ) Yersinia during the exit process at the basolateral side of the Caco-2 cells or ( E ) Yersinia that has successfully egressed from the Caco-2 monolayer are shown; white arrows indicate lamellipodia often associated with the egressing bacteria. ( F ) Caco-2 monolayer was infected with the Y. pseudotuberculosis wildtype strain YPIII (wt), the YP147 (Δ cnfY ) mutant strain +/- the empty vector pJNS11 (pV), or the cnfY + complementation plasmid pJNS10 (p cnfY ). 3 hours post-infection, gentamicin was added to kill extracellular bacteria. After an additional 30 minutes of incubation, the medium from the basolateral chamber was collected and plated on LB plates to determine the CFUs of egressed bacteria. The mean +/- SEM of six independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Šidák correction and indicated by asterisks (P-value: **** < 0.0001). ( G ) Comparative study of bacterial egress of strains with and without a functional cnfY toxin gene. Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of Y. pseudotuberculosis wildtype strain YPIII ( cnfY + ), YP147 (YPIII Δ cnfY ) mutant strain, IP32777 ( cnfY - ), IP32953 ( cnfY - ), IP2666 ( cnfY + ), and the IP2666 Δ cnfY mutant strain. 3 hours post-infection, gentamicin was added to kill extracellular bacteria. After an additional 30 minutes of incubation, the medium from the basolateral chamber was collected and plated onto LB agar to determine the CFUs of egressed bacteria. The mean +/- SEM of four independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001).

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Infection, Bacteria, Mutagenesis, Transmission Assay, Electron Microscopy, Plasmid Preparation, Incubation, Functional Assay

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), the YP147 (YPIII Δ cnfY ) +/- the empty vector pJNS11 (pV), and the cnfY + complementation plasmid pJNS10 (p cnfY ). ( A , D , E ) 3 hours post-infection the medium was removed and replaced by medium containing gentamicin and inhibitors of the Cdc42/Rac1 (ML141) ( A ), phospholipase C (PLC γ-1) (U-73122) ( D ), and Ca 2+ channel IP3-R (2-APB) ( D ), or activators for Cdc42/Rac1 (Activator II), PLC γ-1 (m-3M3FBS), or IP3-R (Myo-Inositol) ( E ). After 30 minutes, the medium from the basolateral chamber was collected and plated onto LB agar to determine the CFUs of egressed bacteria. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001). ( B , C ) Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), the Yersinia virulence-negative strain (ΔpYV), the cnfY -negative mutant strain YP147 (Δ cnfY ) without or with cnfY + complementation plasmid pJNS10 (p cnfY ). After 3.5 h post-infection, infected Caco-2 cells were lysed, cell extracts were prepared. Activation of the different Rho GTPases was tested by the isolation of the GTP-bound form by pull-downs with Rho-GTPase-binding agarose beads and Western blotting using Rho GTPase-specific antibodies, e.g., against Cdc42. M: Protein size marker. As negative and positive controls, high concentrations of GDP and GTP (GTP γS) were added to the uninfected whole-cell extract samples. Equal concentrations of extracts were used for pull-down assays, which were assessed by Western blotting with Actin antibodies. ( B ) shows a scheme of the procedure (Created in BioRender. Dersch, P. (2026)), and ( C ) the Western blots; ( F , G ) CNF Y -mediated induction of inositol triphosphate (IP 3 ) production. ( F ) Scheme of triggered Cdc42-mediated activation of phospholipase C (PLC γ-1), which leads to the formation of inositol monophosphate (IP 3 ) from PIP 2. IP 3 is rapidly metabolized to inositol monophosphate (IP 1 ), and IP 1 can thus be used as a proxy for IP 3 levels by adding LiCl, which blocks the metabolism of IP 1 . Created in BioRender. Dersch, P. (2026). ( G ) Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), YP147 (YPIII Δ cnfY ) +/- the empty vector pJNS11 (pV), and the cnfY + complementation plasmid pJNS10 (p cnfY ). After at least 2 h post-infection, the medium was removed and replaced with medium +/- gentamicin and/or 50 mM LiCl to block IP 1 metabolism, as indicated. After an additional 1.5 h, the Caco-2 cells were lysed, and the cellular concentration of IP 1 was determined by ELISA using an anti-IP 1 monoclonal antibody. The IP 3 concentrations were calculated based on the IP 1 amounts. The mean +/- SEM of four independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001).

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Infection, Bacteria, Plasmid Preparation, Mutagenesis, Activation Assay, Isolation, Binding Assay, Western Blot, Marker, Blocking Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A ) Illustration of the VAMP-Stx-SNAP protein interaction for the formation of the SNARE complex initiating exocytotic processes. Created in BioRender. Dersch, P. (2026). ( B - J ) Caco-2 monolayer was infected with 2,5 x 10 6 bacteria of Y. pseudotuberculosis wildtype YPIII pFU92 ( gfp + ). 3.5 h post-infection, the Caco-2 cell monolayers were analyzed by CLSM to detect Yersinia (GFP-green), the cell plasma membranes (CellMask Plasma Membrane Red-CMPR), the nuclei (DAPI-blue), and SNARE proteins (yellow) colocalized with the bacteria using antibodies against VAMP3 ( B ), VAMP7 ( C,D ), Stx3 ( E ), Stx4 ( F,G ), SNAP25 ( H ), and SNAP23 ( I,J ). Orthogonal XZ and YZ planes are shown.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Infection, Bacteria, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A-D ) Caco-2, and the isogenic cell lines Caco-2ΔStx3, Caco-2ΔStx4, Caco-2ΔSNAP23, and were seeded into transwells and cultivated until the TEER had reached > 400 Ω•cm². Whole-cell extracts were prepared and analyzed by Western blotting using antibodies specific to Stx3 ( A ), Stx4 ( B ), and SNAP23 ( C ), and actin as a loading control ( A-C ). M: protein marker. ( D ) Monolayers of wildtype Caco-2 cells (wt), and ΔStx3, ΔStx4, and ΔSNAP23 mutant derivatives were infected with Y. pseudotuberculosis wildtype strain YPIII ( cnfY + ) or YP147 (Δ cnfY ). The CFUs of egressed bacteria were determined 3.5 h after infection. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001). ( E ) Illustration of the CNF Y -triggered activation of Yersinia egress through the induction of the Cdc42-dependent Ca 2+ -regulated exocytosis machinery. Created in BioRender. Dersch, P. (2026). Upon uptake of Y. pseudotuberculosis, the bacteria reside in a membrane-bound vacuole, to which the v-SNARE protein VAMP-3 is initially recruited. Later, during transcytosis, VAMP3 is replaced by VAMP7. The CNF Y toxin is secreted by the intracellular bacteria (indicated by red arrows), leading to the activation of Cdc42 at the cell membrane by the deamidation of Gln61 of the Rho GTPase. This activates PLC-γ1, leading to the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP 2 ), producing inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 interacts with the IP 3 receptor (IP 3 -R) in the endoplasmic reticulum (ER), which triggers the activation of adjacent Ca 2+ channels and the subsequent formation of a functional SNARE complex of VAMP7, Stx4, and SNAP25, and allows egress of Yersinia by exocytosis.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Western Blot, Control, Marker, Mutagenesis, Infection, Bacteria, Activation Assay, Membrane, Functional Assay

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A ) Illustration of the VAMP-Stx-SNAP protein interaction for the formation of the SNARE complex initiating exocytotic processes. Created in BioRender. Dersch, P. (2026). ( B - J ) Caco-2 monolayer was infected with 2,5 x 10 6 bacteria of Y. pseudotuberculosis wildtype YPIII pFU92 ( gfp + ). 3.5 h post-infection, the Caco-2 cell monolayers were analyzed by CLSM to detect Yersinia (GFP-green), the cell plasma membranes (CellMask Plasma Membrane Red-CMPR), the nuclei (DAPI-blue), and SNARE proteins (yellow) colocalized with the bacteria using antibodies against VAMP3 ( B ), VAMP7 ( C,D ), Stx3 ( E ), Stx4 ( F,G ), SNAP25 ( H ), and SNAP23 ( I,J ). Orthogonal XZ and YZ planes are shown.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Infection, Bacteria, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: ( A-D ) Caco-2, and the isogenic cell lines Caco-2ΔStx3, Caco-2ΔStx4, Caco-2ΔSNAP23, and were seeded into transwells and cultivated until the TEER had reached > 400 Ω•cm². Whole-cell extracts were prepared and analyzed by Western blotting using antibodies specific to Stx3 ( A ), Stx4 ( B ), and SNAP23 ( C ), and actin as a loading control ( A-C ). M: protein marker. ( D ) Monolayers of wildtype Caco-2 cells (wt), and ΔStx3, ΔStx4, and ΔSNAP23 mutant derivatives were infected with Y. pseudotuberculosis wildtype strain YPIII ( cnfY + ) or YP147 (Δ cnfY ). The CFUs of egressed bacteria were determined 3.5 h after infection. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001). ( E ) Illustration of the CNF Y -triggered activation of Yersinia egress through the induction of the Cdc42-dependent Ca 2+ -regulated exocytosis machinery. Created in BioRender. Dersch, P. (2026). Upon uptake of Y. pseudotuberculosis, the bacteria reside in a membrane-bound vacuole, to which the v-SNARE protein VAMP-3 is initially recruited. Later, during transcytosis, VAMP3 is replaced by VAMP7. The CNF Y toxin is secreted by the intracellular bacteria (indicated by red arrows), leading to the activation of Cdc42 at the cell membrane by the deamidation of Gln61 of the Rho GTPase. This activates PLC-γ1, leading to the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP 2 ), producing inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 interacts with the IP 3 receptor (IP 3 -R) in the endoplasmic reticulum (ER), which triggers the activation of adjacent Ca 2+ channels and the subsequent formation of a functional SNARE complex of VAMP7, Stx4, and SNAP25, and allows egress of Yersinia by exocytosis.

Article Snippet: For Caco-2 ΔSNAP23, SNAP23 CRISPR/Cas9 KO Plasmid (h) (Santa Cruz Biotechnology, #sc-417781) and the SNAP23 HDR Plasmid (h) (Santa Cruz Biotechnology, #sc-417781-HDR) were used.

Techniques: Western Blot, Control, Marker, Mutagenesis, Infection, Bacteria, Activation Assay, Membrane, Functional Assay

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution overview. ( A ) SNAP25 sub-family proteins and their isoforms (CLUSTAL 2.1 ). ( B ) Co-crystal structure (PDB: 1XTG ) of LC/A (white) and SNAP25 (dark gray). Eight substitutions in LC/A drive SNAP23 specificity (teal) through substrate control loops (pink) alongside prior substitutions (light gray). ( C ) Platform for the directed evolution of SNAP23 substrate specificity. 1. Random or site-directed mutagenesis (e.g., site-saturation); 2. QC by DNA sequencing; 3. High-throughput protein production; 4. Measure V 0 23 and V 0 25 for substrate specificity; 5. Confirmation screens. The most specific and consistent variant from the DARET assay entered the next round of directed evolution. ( D ) Sequence alignment of substrates used for screening (UniProt: P60880, O00161). The SNAP binding exosites in LC/A (residue numbers above) and cleavage site (scissors) are shown. The gradient of color indicates homology from identical (white, *), to strongly similar (light gray, :), weakly similar (teal, .), or dissimilar (dark teal, space).

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques: Control, Mutagenesis, DNA Sequencing, High Throughput Screening Assay, Variant Assay, Sequencing, Binding Assay, Residue

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Directed evolution of a SNAP23-specific LC/A. ( A ) Improvement in specificity index through directed evolution in salt-free buffer. The most specific variant from each round was assayed multiple times (replicates shown). The average specificity index (horizontal bars) and standard deviation (error bars) are shown. The final dilution of cell lysates for screening is indicated by color code. ( B ) Increased specificity index achieved through directed evolution in salt buffer. ( C ) Roadmap for directed evolution showing the fold increase in specificity indices over eight rounds (R1 to R8). As shown in Fig. S1, the substrate specificity of the qmLC/A variant is sensitive to screening dilution; therefore, each point represents the variant’s average specificity normalized to the qmLC/A average specificity at the corresponding screening dilution. Advancement to the next round (solid line) weighed specificity index, protein solubility, and stability.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques: Variant Assay, Standard Deviation, Solubility

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Enzyme kinetics of LC/A variants.

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques:

Journal: Scientific Reports

Article Title: Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

doi: 10.1038/s41598-022-13617-z

Figure Lengend Snippet: Characterization of omLC/A cleavage. ( A ) Rates of SNAP cleavage by batch-expressed, purified qmLC/A and ( B ) omLC/A at the indicated DARET substrate concentrations (n = 3). ( C ) Deconvoluted ESI–MS of recombinant, full-length SNAP23 (fl-S23) treated with DTT and iodoacetamide to carbamidomethylate its six cysteines (6 × CAM). The mass spectrum of intact fl-S23 incubated with buffer (top, black) is compared to that of fl-S23 incubated with omLC/A (bottom, teal). Intact fl-S23 (1) and cleaved fl-S23 (cl. fl-S23, 2) peaks are labeled. Additional marked peaks correspond to the masses of peaks 1 and 2 plus one additional CAM (+ 57 Da), likely resulting from overalkylation by iodoacetamide . The cleaved peptide was not directly observed, but inferred from the mass difference of peaks 1 and 2. The deconvolution error is +/- 2 Da. ( D ) Recombinant omBoNT/A was purified by IMAC followed by anion exchange (AEX) chromatography. The omBoNT/A is ≈95% nicked upon DTT reduction as demonstrated by the presence of the HC/A and omLC/A bands. ( E ) In vitro cleavage of recombinant fl-S23 by two independent preparations of omBoNT/A (1 and 2) visualized with a C-terminal anti-SNAP23 antibody; before proteolysis, omBoNT/A was reduced with TCEP. The untreated, wtLC/A, and wtLC/E lanes provide negative controls. ( F ) In cellulo cleavage of SNAP23 and SNAP25 in SiMA-H1 cells infected with adenovirus delivering DNA encoding mCherry/SNAP23. Cells were treated with omBoNT/A or wtBoNT/A or without toxin (ct). Proteins, full-length (fl) or cleaved (cl), were identified by Western blotting with anti-SNAP23, -SNAP25, or -mCherry antibodies (M, MW marker). Full-length images of these gels with multiple exposures where necessary are shown in Fig. S12 and S13).

Article Snippet: Human SNAP23 in vitro cleavage was evaluated by incubating 30 μg of human recombinant SNAP23 protein (Novus Biologicals) with 400 nM of either wild-type LC/A, wild type LC/E, or reduced full length omBoNT/A at 37 °C for 1 h in PBS, pH 7.2 (ThermoFisher).

Techniques: Purification, Recombinant, Incubation, Labeling, Chromatography, In Vitro, Infection, Western Blot, Marker