anti-syntaxin4  (Synaptic Systems)

Journal: The Biochemical journal

Article Title: Munc18a Clusters SNARE-Bearing Liposomes Prior to Trans-SNARE Zippering

doi: 10.1042/BCJ20170494

Figure Lengend Snippet: VAMP2-bearing donor liposomes were incubated 3 h on ice with acceptor proteoliposomes as specified, with or without Munc18a. Each sample was then aliquoted for clustering and lipid-mixing assays. A) Cumulative distribution plots show the results of the clustering assay. B) Following the lipid-mixing assay, the mean values of the maximal lipid-mixing rates are presented and error bars indicate SD (n=4). Student’s t-test was used to assess the significance of the difference between the “– Munc18a” results and the “+ Munc18” results. * indicates P < 0.05 whereas ** indicates P < 0.01. Note that the extent of Munc18a-dependent acceleration of lipid mixing does not necessarily correlate linearly with the extent of change in clustering, which implies either the insufficient resolution of the clustering assay or possibly different modes of action exploited by Munc18a in clustering and lipid mixing respectively. C) To determine the biochemical interaction between Munc18a and Q-SNAREs, His6-Munc18a was incubated with specified proteoliposomes at 4°C overnight before Ni-NTA agarose was added to bring down His6-Munc18a and associating molecules. Eluates were subject to SDS-PAGE followed by Coomassie Brilliant Blue staining. The top and bottom panels are from two separate gels. D) Densitometry was performed and the intensity of the syntaxin bands in the pull-down was normalized for the input. The values of the wild-type syntaxin3 and syntaxin4 were set as 100% respectively and then used to calculate the relative levels of mutant syntaxins in the pull-down. Student’s t-test was used to assess the significance of the difference between the “WT” results and the “∆N” results. ** indicates P < 0.01. Error bars represent SD (n=3).

Article Snippet: For immuno-precipitation (IP) experiments described in , overnight fusion reaction (20 μL) on ice was terminated by the addition of 1 μL of anti-syntaxin4 rabbit serum (Antibodies–online; cat#: ABIN1742221), and the incubation was continued on ice for 1 h. Three hundred and eighty μL of RIPA buffer (25 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1% NP40 alternative, 1% deoxycholate, 0.1% SDS, and 10 mM EDTA) were then added and the mixture were nutated at 4°C for 20 min before centrifugation at 16,000 × g for 5 min at 4°C.

Techniques: Incubation, SDS Page, Staining, Mutagenesis

Journal: The Biochemical journal

Article Title: Munc18a Clusters SNARE-Bearing Liposomes Prior to Trans-SNARE Zippering

doi: 10.1042/BCJ20170494

Figure Lengend Snippet: Donor and acceptor proteoliposomes as indicated were incubated 3 h on ice alone or with Munc18a (2μM) that had been preincubated overnight with specified amounts of Stx4-Nwt, Stx4-NL8A, or control buffer. The reaction mixtures were then divided for lipid-mixing assay and clustering assay respectively. A) To assess the potential of N-peptide to stimulate Munc18a function in reactions containing VAMP2-bearing donor liposomes and syntaxin4∆N/SNAP23-bearing acceptor liposomes, the ratio of the maximal lipid-mixing rate of each Munc18a-containing reaction over that of the SNARE-only reaction was calculated and shown as fold of stimulation. B) To assess the potential of N-peptide to inhibit Munc18a activity in reactions containing VAMP2-bearing donor liposomes and syntaxin4/SNAP23-bearing acceptor liposomes, the maximal lipid-mixing rate of the peptide-free reaction (but with Munc18a) was set at 100% and then used to calculate the relative values of the other reactions. Student’s t-test was used in statistical analysis. ** indicates P < 0.01. Error bars indicate SD (n=4). Cumulative distribution plots in A) and B) highlight the impact of the wild-type and mutant N-peptides on membrane clustering.

Article Snippet: For immuno-precipitation (IP) experiments described in , overnight fusion reaction (20 μL) on ice was terminated by the addition of 1 μL of anti-syntaxin4 rabbit serum (Antibodies–online; cat#: ABIN1742221), and the incubation was continued on ice for 1 h. Three hundred and eighty μL of RIPA buffer (25 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1% NP40 alternative, 1% deoxycholate, 0.1% SDS, and 10 mM EDTA) were then added and the mixture were nutated at 4°C for 20 min before centrifugation at 16,000 × g for 5 min at 4°C.

Techniques: Incubation, Activity Assay, Mutagenesis

Journal: The Biochemical journal

Article Title: Munc18a Clusters SNARE-Bearing Liposomes Prior to Trans-SNARE Zippering

doi: 10.1042/BCJ20170494

Figure Lengend Snippet: A) Incubation procedure. Syntaxin4/His6-SNAP23-bearing acceptor liposomes were incubated with either the soluble, inhibitory VAMPs (10-fold molar excess in comparison to the membrane-bound VAMP2 added next) or buffer for 1 h on ice before receiving the VAMP2-bearing donor liposomes and Munc18a. Following another 15 h incubation on ice, samples were aliquoted and subject to B) lipid-mixing assay, C) pull-down assays using Ni-NTA resin, D) co-immunoprecipitation using immobilized anti-Stx4 antibody, and E) clustering assay. Student’s t-test was used to assess the statistical difference between the results in lane 3 and the rest in B). * indicates P < 0.05. Error bars indicate SD (n=3).

Article Snippet: For immuno-precipitation (IP) experiments described in , overnight fusion reaction (20 μL) on ice was terminated by the addition of 1 μL of anti-syntaxin4 rabbit serum (Antibodies–online; cat#: ABIN1742221), and the incubation was continued on ice for 1 h. Three hundred and eighty μL of RIPA buffer (25 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1% NP40 alternative, 1% deoxycholate, 0.1% SDS, and 10 mM EDTA) were then added and the mixture were nutated at 4°C for 20 min before centrifugation at 16,000 × g for 5 min at 4°C.

Techniques: Incubation, Immunoprecipitation