rabbit anti syntaxin4  (Alomone Labs)

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

Journal: Scientific Reports

Article Title: CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca 2+ -independent manner via molecular interaction with syntaxin4

doi: 10.1038/srep28133

Figure Lengend Snippet: ( a) CRACM3 and syntaxin4 protein levels were measured by western blotting after immunoprecipitation using specific antibodies for CRACM1, CRACM2, CRACM3, and syntaxin4. Full-length blots are presented in . Upper: expression of CRACM3 in TG-stimulated cells (lane 1–4). Lower: expression of syntaxin4 in TG-stimulated cells (lane 1–4). (b) In situ PLA assay in non-stimulated cell and TG-stimulated cells. TG-stimulated or non-stimulated RBL-2H3 cells were used for in situ PLA analysis using a Duolink In Situ Detection Reagents Far Red detection kit. The slides were examined by confocal microscopy. The imaging data were analysed with Duolink ImageTool. Upper: Red PLA fluorescence signal, phalloidin green fluorescence signal, DAPI fluorescence signal and merged image in non-stimulated control cells. Lower: Red PLA fluorescence signal, phalloidin green fluorescence signal, DAPI fluorescence signal and merged image in TG-stimulated control cells. ( c) Positive PLA area in non-stimulated and TG-stimulated cells normalized to DAPI fluorescence (n = 42, *** P < 0.001 ). ( d) The expression of CRACM3 and syntaxin4 at 0 min, 1 min, 5 min, 15 min in TG-stimulated cells detected by confocal microscopy. ( e) Normalized fluorescence signals of CRACM3 and syntaxin4 using DAPI fluorescence (n = 40, * P < 0.05). Cells were examined by confocal microscopy, and imaging data were analysed with ImageJ software.

Article Snippet: After being blocked with 3% bovine serum albumin in phosphate-buffered saline, the primary antibodies rabbit polyclonal anti-syntaxin4 IgG (SIGMA-ALDRICH, St. Louis, MO, USA) and monoclonal anti-CRACM3 IgG (clone 2H2G9, Abnova, Taiwan) were added to the slides.

Techniques: Western Blot, Immunoprecipitation, Expressing, In Situ, Confocal Microscopy, Imaging, Fluorescence, Software

Journal: Scientific Reports

Article Title: CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca 2+ -independent manner via molecular interaction with syntaxin4

doi: 10.1038/srep28133

Figure Lengend Snippet: All plasmids were transfected into cells using RBL-2H3 Cell Avalanche TM Transfection Reagent. At 72 h after transfection, the cells were stimulated with 0.5 μM TG for 15 min, and His-tag immunoprecipitation followed by western blotting using specific antibodies was performed. ( a) CRACM3 protein, which was co-immunoprecipitated using the His-tag in rSx4-His-wild- (lane 1), rSx4-His-mTM- (lane 2), rSx4-His-mH3- (lane 3), rSx4-His-mIIS- (lane 4), and rSx4-His-mNp-transfected cells (lane 5), was detected using western blotting. Full-length blots are presented in . Upper: CRACM3 expression in plasmid-transfected cells. Lower: CRACM3 expression normalized against actin levels in total cell lysates. ( b) The protein levels of CRACM3 in His-tagged CRACM3 truncation-transfected cells after His-tag immunoprecipitation. (a.u., arbitrary area units; *** P < 0.001 ; n = 3). ( c) Syntaxin4 protein, which was co-immunoprecipitated with His-tag in rM3-His wild- (lane 1), rM3-His-mNTD- (lane 2), rM3-His-mL1- (lane 3), rM-His-mL2- (lane 4), rM3-His-mL3- (lane 5), and rM3-His-mCTD-transfected cells (lane 6), was detected by western blotting. Full-length blots are presented in .Upper: syntaxin4 expression in the plasmid-transfected cells. Lower: syntaxin4 expression normalized against actin levels in total cell lysates. ( d) The protein levels of syntaxin4 in His-tagged CRACM3 truncation-transfected cells after His-tag immunoprecipitation. (a.u., arbitrary units; *** P < 0.001 ; n = 3).

Article Snippet: After being blocked with 3% bovine serum albumin in phosphate-buffered saline, the primary antibodies rabbit polyclonal anti-syntaxin4 IgG (SIGMA-ALDRICH, St. Louis, MO, USA) and monoclonal anti-CRACM3 IgG (clone 2H2G9, Abnova, Taiwan) were added to the slides.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca 2+ -independent manner via molecular interaction with syntaxin4

doi: 10.1038/srep28133

Figure Lengend Snippet: (a) The protein levels of CRACM3 and syntaxin4 3 d post transfection of siRNA were detected by western blotting following siRNA-mediated protein suppression. Upper: CRACM3 expression in NCsiRNA- (lane 1), Syn4siRNA- (lane 2), and M3siRNA-transfected cells (lane 3). Middle: syntaxin4 expression in each group of cells. Lower: actin expression in each group of cells. (b) CRACM3 and syntaxin4 expression normalized against actin levels (a.u., arbitrary units; *** P < 0.001 ; n = 3). The samples derive from the same experiment and that blots were processed in parallel. (c) Overexpression of CRACM3 was observed in rM-wild-transfected RBL-2H3 cells. CRACM3 expression was detected by western blotting 10 d post transfection. Upper: CRACM3 expression in total cells lysates of control cells (lane 1), rM3-His-wild-transfected cells (lane 2), and rM3-wild-transfected cells (lane 3). Middle: CRACM3 expression detected by western blotting followed by syntaxin4 immunoprecipitation in each group of cells. Lower: actin expression in the total cell lysates of each group of cells. (d) CRACM3 expression in total cells lysates and after syntaxin4 immunoprecipitation normalized against actin levels (a.u., arbitrary unit; * P < 0.05 ; *** P < 0.001 ; n = 3). The samples derive from the same experiment and that blots were processed in parallel. (e) Typical Ca 2+ influx patterns in cameleon (YC4.60)-transfected cells. The assay was performed using a fluorometric imaging plate reader, and the results are presented as the ratio of relative fluorescence units (R.F.U. 530 nm/480 nm). (f) Extracellular Ca 2+ titration curve of M3siRNA-transfected, NCsiRNA-transfected, and rM3-wild-transfected cells (n = 8–10) that were co-transfected with YC4.60. The data are presented as the means ± s.e.m. (g) A typical C m curve was measured under whole-cell clamp conditions, and the change in C m was triggered by 3 μM [Ca 2+ ] free. (h) The [Ca 2+ ] i -dependence of the capacitance responses (Δ Cm ) was observed in M3siRNA-transfected, NCsiRNA-transfected, and rM3-wild-transfected cells (* P < 0.05 ; n = 25–40 individual cells per group). The data are presented as the means ± s.e.m. (i) The level of released histamine in control cells, NCsiRNA-transfected cells, M3siRNA-transfected cells, and rM3-wild-transfected cells (* P < 0.05 ; *** P < 0.001 ; n = 6). The results are expressed as the mean ± s.e.m. All full-length blots are presented in .

Article Snippet: After being blocked with 3% bovine serum albumin in phosphate-buffered saline, the primary antibodies rabbit polyclonal anti-syntaxin4 IgG (SIGMA-ALDRICH, St. Louis, MO, USA) and monoclonal anti-CRACM3 IgG (clone 2H2G9, Abnova, Taiwan) were added to the slides.

Techniques: Transfection, Western Blot, Expressing, Over Expression, Immunoprecipitation, Imaging, Fluorescence, Titration

Journal: Scientific Reports

Article Title: CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca 2+ -independent manner via molecular interaction with syntaxin4

doi: 10.1038/srep28133

Figure Lengend Snippet: The sequences of siRNA.

Article Snippet: After being blocked with 3% bovine serum albumin in phosphate-buffered saline, the primary antibodies rabbit polyclonal anti-syntaxin4 IgG (SIGMA-ALDRICH, St. Louis, MO, USA) and monoclonal anti-CRACM3 IgG (clone 2H2G9, Abnova, Taiwan) were added to the slides.

Techniques: Negative Control