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Vamp 2

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VAMP-2 is a recombinant protein that functions as a vesicle-associated membrane protein. It is a component of the SNARE complex involved in the docking and fusion of synaptic vesicles with the presynaptic membrane.

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Lab products found in correlation

Snap 25, by Fortrea (2 mentions) Syntaxin, by Merck Group (1 mentions) Neuronal nuclei (neun), by Merck Group (1 mentions) βiii tubulin, by Fortrea (1 mentions) Snap 25, by BD (1 mentions)

3 protocols using vamp 2

1

Western Blot Analysis of BoNT-Intoxicated Neurons

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For the western blot analysis shown in Fig 1, fully differentiated mouse ES-MNs were treated with the compounds (20 μM) (Fig 2) 30 min prior to the addition of 250pM BoNT/A. For hES-MN pre-intoxication models shown in Figs 3A, 4A and 5A, fully differentiated human ES-MNs (Day 35) were treated with the titrated concentrations of compounds for 30 min and then intoxicated with either BoNT/A, BoNT/B or trypsin-activated BoNT/E (MetaBiologics, Madison, WI), and incubated at 37°C for 4 hr. Bafilomycin was utilized as a positive control [30 (link)]. For post-intoxication studies, cells were intoxicated with either BoNT/A, BoNT/B or trypsin-activated BoNT/E for either 30 or 60 min and then 20 μM compound was added to the cultures. Total intoxication time was kept constant at 4 hr for all immunoblotting analyses. Following intoxication, samples were prepared and BoNT mediated SNARE protein cleavage was quantified using standard immunoblotting procedures employing SNAP-25 (Covance) and VAMP-2 (R&D Systems) antibodies—as described previously [30 (link), 36 (link), 37 (link)]. The reported values were calculated from at least three independent experiments.
Kiris E., Nuss J.E., Stanford S.M., Wanner L.M., Cazares L., Maestre M.F., Du H.T., Gomba G.Y., Burnett J.C., Gussio R., Bottini N., Panchal R.G., Kane C.D., Tessarollo L, & Bavari S. (2015). Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays. PLoS ONE, 10(6), e0129264.
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2

Quantifying BoNT Cleavage Activity in hES-MN Cultures

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hES-MN cultures (day 35) were intoxicated with increasing concentrations of either BoNT/A or BoNT/B or trypsin-activated BoNT/E (MetaBiologics) and incubated at 37 °C for 4 h (Fig. 3). Following intoxication, samples were processed, and SNAP-25 (Covance) and VAMP-2 (R&D Systems) protein cleavages were quantified using standard immunoblotting procedures—as described previously (Huang et al. 2011 (link); Pellett et al. 2007 (link)). Quantification of changes in total VAMP-2 protein levels was calculated by normalizing the total VAMP-2 band intensity values to corresponding GAPDH levels relative to non-toxin treated control conditions run on each gel. For inhibitor studies, Triticium vulgaris Lectin (TVL) and bafilomycin were used at titrated concentrations and added to the cultures 30 min prior to intoxication. Both reagents were obtained from Sigma. An antibody that neutralizes BoNT/A (4A2-4) (produced at the US Army Medical Research Institute of Infectious Diseases) and the control antibody (Anti staphylococcal enterotoxin B) (Toxin Technology) were simultaneously applied with 1 nM BoNT/A to the cultures.
Kiris E., Burnett J.C., Nuss J.E., Wanner L.M., Peyser B.D., Du H.T., Gomba G.Y., Kota K.P., Panchal R.G., Gussio R., Kane C.D., Tessarollo L, & Bavari S. (2015). Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons. Neurotoxicity research, 27(4), 384-398.
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Immunocytochemical Characterization of hES-MN Cultures

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hES-MN cultures (day 35) were fixed in 4 % paraformaldehyde-PBS for 10 min and permeabilized with 0.1 % Triton X-100. After being blocked with 10 % serum, the cells were then incubated overnight with the following primary antibodies: Tau5 (Thermo Scientific), LIM3 (Millipore), ChAT (Millipore), NeUN (Millipore), Hb9 [Developmental Studies Hybridoma Bank (DSHB)], Isl1 (DSHB), and β-III tubulin (Covance) (as shown in Fig. 1b), as well as antibodies for SNAP-25 (BD Biosciences), SV2A (Millipore), Syntaxin (Sigma), VAMP-2 (R&D Systems), and MAP2 (Millipore) (as shown in Fig. 2b) in PBS with 10 % serum according to the manufacturers’ suggested working concentrations. On the following day, appropriate secondary antibodies, conjugated with Alexa488 and Alexa594, were incubated with the cells for 2 h at room temperature. Image acquisition was performed using Zeiss or Opera (PerkinElmer) confocal microscopes.
Kiris E., Burnett J.C., Nuss J.E., Wanner L.M., Peyser B.D., Du H.T., Gomba G.Y., Kota K.P., Panchal R.G., Gussio R., Kane C.D., Tessarollo L, & Bavari S. (2015). Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons. Neurotoxicity research, 27(4), 384-398.
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