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Addgene inc rat syntaxin3

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 <t>syntaxin3</t> 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).

Rat Syntaxin3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Munc18a Clusters SNARE-Bearing Liposomes Prior to Trans-SNARE Zippering"

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

Journal: The Biochemical journal

doi: 10.1042/BCJ20170494

Figure Legend 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).

Techniques Used: Liposomes, Incubation, SDS Page, Staining, Mutagenesis

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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: The cDNAs for full-length or N-terminally truncated rat syntaxin3 and syntaxin4 were inserted individually into the LIC site of pET MBP His 6 LIC cloning vector (gift from Scott Gradia; Addgene plasmid # 37237) to generate pET-Stx3-TCS(Tev Cleavable Site)-MBP-His 6 , pET-Stx3ΔN-TCS-MBP-His 6 , pET-Stx4-TCS-MBP-His 6 , and pET-Stx4ΔN-TCS-MBP-His 6 .

Techniques: Liposomes, Incubation, SDS Page, Staining, Mutagenesis

Munc13-4 binds to syntaxin 7 and VAMP8 in a calcium-dependent manner. (A) Binding assays for Munc13-4 and SNARE proteins. The binding of Flag-Munc13-4 to several GFP-tagged SNARE proteins or GFP (E. Vec) was analyzed by the TR-FRET assay as described in Materials and Methods . The reactions were carried out using 293T cell lysates. Where indicated, the reactions were performed in the presence of 100 μM CaCl 2 or 200 μM EGTA. Triplicates of one experiment, representative of at least four experiments. * p < 0.05; NS, not significant. STX, syntaxin. (B) Binding assays of Munc13-4 and syntaxin 7 in the presence of 100 μM EGTA or CaCl 2 at the indicated total calcium concentration. Emission ratios were normalized to sample NA (no addition). The results are expressed as mean ± SEM. NS, not significant. *** p < 0.001 and * p < 0.05 vs. EGTA. (C, D) Coimmunoprecipitation assays were carried out using anti-Flag beads and 293T cell lysates expressing the indicated proteins. The cells were disrupted by nitrogen cavitation in either relaxation buffer (C) or RIPA buffer (D) as described in Materials and Methods . Western blots are representative of at least three experiments with similar results. The asterisk indicates truncations. (E, F) Analysis of the binding of Munc13-4 WT or mutants C2A*, C2B*, and C2A*C2B* to syntaxin 7 (E) or VAMP8 (F). C2A* includes point mutations D127 and D133 to alanines in the Munc13-4 C2A domain, which knocks out the Ca 2+ -binding sites in this domain; C2B* includes point mutations D941 and D947 to alanines to knock out the Ca 2+ -binding sites in the C2B domain. C2A*C2B* includes four D → A mutations corresponding to both C2 domains. NS, not significant. ** p < 0.001. (G) Coimmunoprecipitation assays were performed as in C, except that reactions were performed in the presence or absence of 100 μM calcium (Ca 2+ ), and, where indicated, Munc13-4 C2 mutants were used instead of WT Munc13-4.

Journal: Molecular Biology of the Cell

Article Title: Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

doi: 10.1091/mbc.E15-05-0283

Figure Lengend Snippet: Munc13-4 binds to syntaxin 7 and VAMP8 in a calcium-dependent manner. (A) Binding assays for Munc13-4 and SNARE proteins. The binding of Flag-Munc13-4 to several GFP-tagged SNARE proteins or GFP (E. Vec) was analyzed by the TR-FRET assay as described in Materials and Methods . The reactions were carried out using 293T cell lysates. Where indicated, the reactions were performed in the presence of 100 μM CaCl 2 or 200 μM EGTA. Triplicates of one experiment, representative of at least four experiments. * p < 0.05; NS, not significant. STX, syntaxin. (B) Binding assays of Munc13-4 and syntaxin 7 in the presence of 100 μM EGTA or CaCl 2 at the indicated total calcium concentration. Emission ratios were normalized to sample NA (no addition). The results are expressed as mean ± SEM. NS, not significant. *** p < 0.001 and * p < 0.05 vs. EGTA. (C, D) Coimmunoprecipitation assays were carried out using anti-Flag beads and 293T cell lysates expressing the indicated proteins. The cells were disrupted by nitrogen cavitation in either relaxation buffer (C) or RIPA buffer (D) as described in Materials and Methods . Western blots are representative of at least three experiments with similar results. The asterisk indicates truncations. (E, F) Analysis of the binding of Munc13-4 WT or mutants C2A*, C2B*, and C2A*C2B* to syntaxin 7 (E) or VAMP8 (F). C2A* includes point mutations D127 and D133 to alanines in the Munc13-4 C2A domain, which knocks out the Ca 2+ -binding sites in this domain; C2B* includes point mutations D941 and D947 to alanines to knock out the Ca 2+ -binding sites in the C2B domain. C2A*C2B* includes four D → A mutations corresponding to both C2 domains. NS, not significant. ** p < 0.001. (G) Coimmunoprecipitation assays were performed as in C, except that reactions were performed in the presence or absence of 100 μM calcium (Ca 2+ ), and, where indicated, Munc13-4 C2 mutants were used instead of WT Munc13-4.

Article Snippet: Histidine-tagged syntaxin 3 and syntaxin 4 were obtained from Addgene and then subcloned into the EGFP-C1 vector (Clontech Laboratories, Mountain View, CA). mCherry-Munc13-4 and flag-Munc13-4 were obtained from Genecopoeia (Rockville, MD).

Techniques: Binding Assay, Concentration Assay, Expressing, Western Blot, Knock-Out

Syntaxin 7 and Munc13-4 colocalize in LAMP1-positive granules in neutrophils, but the subcellular localization of syntaxin 7 and VAMP8 is independent of Munc13-4. (A) Immunofluorescence analysis of endogenous syntaxin 7 (STX7; green), the late endosomal marker LAMP1 (red), and Munc13-4 (violet) in wild-type neutrophils. Arrowheads indicate examples of vesicles that are positive for all three stainings. Cell nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). (B) Immunofluorescence analysis of endogenous syntaxin 7 (green), the lysosome-related organelle marker MPO (red), and Munc13-4 (violet) in wild-type neutrophils. (C) Colocalization analysis of LAMP1 with either syntaxin 7 or Munc13-4 using 29 and 38 cells, respectively. (D, E) High-resolution STORM analysis of the localization of endogenous Munc13-4 and syntaxin 7 (D) or LAMP1 (E) in wild-type cells. Whereas syntaxin 7 and Munc13-4 are detected adjacent to each other (arrowheads, 10–50 nm; D), LAMP1 and Munc13-4 are in close proximity but not always adjacent (arrowheads, estimated distance >50 nm; E). Scale bar, 1 μm. (F) TIRFM analysis of colocalization of syntaxin 7 and Munc13-4 in RPE cells. Scale bar, 10 μm. (G, H) Immunofluorescence analyses of endogenous syntaxin 7 (green) and VAMP8 (violet) with LAMP1 (red; G) or MPO (red; H) in wild-type and Munc13-4–KO neutrophils. Arrowheads indicate examples of vesicles that are positive for all three stainings. Scale bar, 1 μm. (I) Quantification of the colocalization shown in G and H. Calculation of the colocalization coefficient was performed by analyzing at least 38 cells in each group. Results are mean ± SEM. *** p < 0.001.

Journal: Molecular Biology of the Cell

Article Title: Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

doi: 10.1091/mbc.E15-05-0283

Figure Lengend Snippet: Syntaxin 7 and Munc13-4 colocalize in LAMP1-positive granules in neutrophils, but the subcellular localization of syntaxin 7 and VAMP8 is independent of Munc13-4. (A) Immunofluorescence analysis of endogenous syntaxin 7 (STX7; green), the late endosomal marker LAMP1 (red), and Munc13-4 (violet) in wild-type neutrophils. Arrowheads indicate examples of vesicles that are positive for all three stainings. Cell nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). (B) Immunofluorescence analysis of endogenous syntaxin 7 (green), the lysosome-related organelle marker MPO (red), and Munc13-4 (violet) in wild-type neutrophils. (C) Colocalization analysis of LAMP1 with either syntaxin 7 or Munc13-4 using 29 and 38 cells, respectively. (D, E) High-resolution STORM analysis of the localization of endogenous Munc13-4 and syntaxin 7 (D) or LAMP1 (E) in wild-type cells. Whereas syntaxin 7 and Munc13-4 are detected adjacent to each other (arrowheads, 10–50 nm; D), LAMP1 and Munc13-4 are in close proximity but not always adjacent (arrowheads, estimated distance >50 nm; E). Scale bar, 1 μm. (F) TIRFM analysis of colocalization of syntaxin 7 and Munc13-4 in RPE cells. Scale bar, 10 μm. (G, H) Immunofluorescence analyses of endogenous syntaxin 7 (green) and VAMP8 (violet) with LAMP1 (red; G) or MPO (red; H) in wild-type and Munc13-4–KO neutrophils. Arrowheads indicate examples of vesicles that are positive for all three stainings. Scale bar, 1 μm. (I) Quantification of the colocalization shown in G and H. Calculation of the colocalization coefficient was performed by analyzing at least 38 cells in each group. Results are mean ± SEM. *** p < 0.001.

Techniques: Immunofluorescence, Marker, Staining

Munc13-4 wild type but not the syntaxin 7 binding–deficient mutant Munc13-4 C2A*C2B* rescues the late endosome enlargement phenotype in Munc13-4–KO cells. (A) WT and Munc13-4–KO ( Jinx ) neutrophils were cotransfected with EGFP-LAMP1 and mCherry-Munc13-4 wild type (Munc13-4 WT), mCherry-Munc13-4 C2A*C2B* mutant (Munc13-4C2A*C2B*), or mCherry empty vector (E. Vec). Quantification of EGFP-LAMP1–positive late endosome diameters was performed as in . Results are mean ± SEMs, with 40, 47, 32, and 42 cells quantified for groups WT with mCherry empty vector, Jinx with mCherry empty vector, Jinx with mCherry-Munc13-4-WT, and Jinx with mCherry-Munc13-4-C2A*C2B* mutant, respectively. *** p < 0.0001. (B) Representative TIRFM images of cells from A. Cells were imaged 6 h after nucleofection. Scale bar, 1 μm.

Journal: Molecular Biology of the Cell

Article Title: Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

doi: 10.1091/mbc.E15-05-0283

Figure Lengend Snippet: Munc13-4 wild type but not the syntaxin 7 binding–deficient mutant Munc13-4 C2A*C2B* rescues the late endosome enlargement phenotype in Munc13-4–KO cells. (A) WT and Munc13-4–KO ( Jinx ) neutrophils were cotransfected with EGFP-LAMP1 and mCherry-Munc13-4 wild type (Munc13-4 WT), mCherry-Munc13-4 C2A*C2B* mutant (Munc13-4C2A*C2B*), or mCherry empty vector (E. Vec). Quantification of EGFP-LAMP1–positive late endosome diameters was performed as in . Results are mean ± SEMs, with 40, 47, 32, and 42 cells quantified for groups WT with mCherry empty vector, Jinx with mCherry empty vector, Jinx with mCherry-Munc13-4-WT, and Jinx with mCherry-Munc13-4-C2A*C2B* mutant, respectively. *** p < 0.0001. (B) Representative TIRFM images of cells from A. Cells were imaged 6 h after nucleofection. Scale bar, 1 μm.

Techniques: Binding Assay, Mutagenesis, Plasmid Preparation

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