Review

syntaxin 1a (OriGene)

OriGene is a verified supplier

OriGene manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

OriGene syntaxin 1a

Figure 1. Schematics of the proteins involved in this study. (a) Molecular models of SNAP-25 (based on PDB: 1KIL, in red) and <t>Syntaxin</t> <t>1A</t> (based on PDBs: 1HVV & 1BR0, in green) residing in the plasma membrane (in yellow). On the right we show a nanobody (PDB: 1I3V, in purple) bearing a single Atto647N at the C-terminus, and a complex of a primary and a randomly-labeled secondary antibody with Atto647N on lysines (PDB: 1IGY in blue and light-blue). All molecular models are displayed in the same scale and the bar represents 2 nm. (b) Schematic view of antigens used for the immunization and their wild-type forms. Important functional domains are marked in gray (TM = trans-membrane domain) and the amino acids positions are denoted below. For immunization (injected), all four cysteine residues of SNAP-25 were mutated to serines. For injection of Syntaxin 1A, only the cytosolic portion of the molecule was used to facilitate it expression in E. coli.

Syntaxin 1a, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/syntaxin/Syntaxin+1a+(STX1A)+(NM_001165903)+Human+Untagged+Clone/pm30466346-259-5-11
Average 90 stars, based on 1 article reviews

syntaxin 1a - by Bioz Stars, 2026-11

90/100 stars

Images

1) Product Images from "Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons."

Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons.

Journal: mAbs

doi: 10.1080/19420862.2018.1551675

Figure 1. Schematics of the proteins involved in this study. (a) Molecular models of SNAP-25 (based on PDB: 1KIL, in red) and Syntaxin 1A (based on PDBs: 1HVV & 1BR0, in green) residing in the plasma membrane (in yellow). On the right we show a nanobody (PDB: 1I3V, in purple) bearing a single Atto647N at the C-terminus, and a complex of a primary and a randomly-labeled secondary antibody with Atto647N on lysines (PDB: 1IGY in blue and light-blue). All molecular models are displayed in the same scale and the bar represents 2 nm. (b) Schematic view of antigens used for the immunization and their wild-type forms. Important functional domains are marked in gray (TM = trans-membrane domain) and the amino acids positions are denoted below. For immunization (injected), all four cysteine residues of SNAP-25 were mutated to serines. For injection of Syntaxin 1A, only the cytosolic portion of the molecule was used to facilitate it expression in E. coli.

Figure Legend Snippet: Figure 1. Schematics of the proteins involved in this study. (a) Molecular models of SNAP-25 (based on PDB: 1KIL, in red) and Syntaxin 1A (based on PDBs: 1HVV & 1BR0, in green) residing in the plasma membrane (in yellow). On the right we show a nanobody (PDB: 1I3V, in purple) bearing a single Atto647N at the C-terminus, and a complex of a primary and a randomly-labeled secondary antibody with Atto647N on lysines (PDB: 1IGY in blue and light-blue). All molecular models are displayed in the same scale and the bar represents 2 nm. (b) Schematic view of antigens used for the immunization and their wild-type forms. Important functional domains are marked in gray (TM = trans-membrane domain) and the amino acids positions are denoted below. For immunization (injected), all four cysteine residues of SNAP-25 were mutated to serines. For injection of Syntaxin 1A, only the cytosolic portion of the molecule was used to facilitate it expression in E. coli.

Techniques Used: Clinical Proteomics, Membrane, Labeling, Functional Assay, Injection, Expressing

Figure 3. Specificity test for the selected nanobodies in immunofluorescence and Western Blots. (a & b) COS-7 cells transiently transfected with SNAP-25-EGFP or Syntaxin 1A-EGFP were stained with S25-Nb10 or Stx1A-Nb6 conjugated with a single Atto647N fluorophore. The nanobody signal correlates with the EGFP signal and shows no staining in untransfected cells (revealed by Hoechst-nuclear staining; white arrowheads). The scale bar represents 10 µm. Note that Syntaxin 1A accumulates in the ER-Golgi region due to an impaired export caused by the lack of neuronal cofactors, as it was found in the past30 (c & d) The following samples were loaded in a denaturating SDS-PAGE and were blotted on a nitrocellulose membrane (2 µg of each purified protein, and 20 µg of total protein for the cell or brain lysates): E. coli-purified full length SNAP-25 including a Twin-Strep-Tag (tst; 27.8 kDa), or Syntaxin 1A fused to a SUMO domain and a Twin-Strep-Tag (44.7 kDa); lysates from HEK293 cells transiently transfected with full length Syntaxin 1A-tst (HEK-Stx1A, 37.5 kDa) or SNAP-25-tst (HEK-SNAP-25, 27.8 kDa); whole rat brain and rat primary hippocampal neurons (15 days in vitro). Detection was performed using S25-Nb10 (c) or Stx1A-Nb6 (d) conjugated to a single Atto647N. Both candidates specifically detect the bands at the expected molecular weights (displayed in the protein schematics), while showing no cross-reactivity to the opposite antigen or to any other proteins present in the lysates.

Figure Legend Snippet: Figure 3. Specificity test for the selected nanobodies in immunofluorescence and Western Blots. (a & b) COS-7 cells transiently transfected with SNAP-25-EGFP or Syntaxin 1A-EGFP were stained with S25-Nb10 or Stx1A-Nb6 conjugated with a single Atto647N fluorophore. The nanobody signal correlates with the EGFP signal and shows no staining in untransfected cells (revealed by Hoechst-nuclear staining; white arrowheads). The scale bar represents 10 µm. Note that Syntaxin 1A accumulates in the ER-Golgi region due to an impaired export caused by the lack of neuronal cofactors, as it was found in the past30 (c & d) The following samples were loaded in a denaturating SDS-PAGE and were blotted on a nitrocellulose membrane (2 µg of each purified protein, and 20 µg of total protein for the cell or brain lysates): E. coli-purified full length SNAP-25 including a Twin-Strep-Tag (tst; 27.8 kDa), or Syntaxin 1A fused to a SUMO domain and a Twin-Strep-Tag (44.7 kDa); lysates from HEK293 cells transiently transfected with full length Syntaxin 1A-tst (HEK-Stx1A, 37.5 kDa) or SNAP-25-tst (HEK-SNAP-25, 27.8 kDa); whole rat brain and rat primary hippocampal neurons (15 days in vitro). Detection was performed using S25-Nb10 (c) or Stx1A-Nb6 (d) conjugated to a single Atto647N. Both candidates specifically detect the bands at the expected molecular weights (displayed in the protein schematics), while showing no cross-reactivity to the opposite antigen or to any other proteins present in the lysates.

Techniques Used: Immunofluorescence, Western Blot, Transfection, Staining, SDS Page, Membrane, Purification, Strep-tag, In Vitro

Figure 4. Both nanobodies reveled epitopes that are not reported by antibodies in highly crowded regions of endogenously-expressing PC12 cells or over-expressing COS-7 cells. (a) PC12 cells were co-stained with conventional monoclonal antibodies and with our fluorescently labeled nanobodies. Monoclonal anti SNAP-25 and Syntaxin 1A (clone 71.1 and HPC-1) were detected with a secondary antibody conjugated to Abberior-Star580 (in green). S25-Nb10 or Stx1A-Nb6 were conjugated to a single Atto647N fluorophore (in red). Laser scanning confocal images of the nanobody and the antibody signals colocalize relatively good at the plasma membrane, but the nanobodies also reveal stronger signals in the perinuclear areas (especially evident for SNAP-25). The scale bar represents 10 µm. (b) Laser scanning confocal images of COS-7 cells transiently transfected with SNAP-25 or Syntaxin 1A fused to EGFP (in green) and co-stained with monoclonal primary and Cy3-fluorescently labeled secondary antibody (clone 71.1 and clone 78.2, in magenta) and nanobodies directly conjugated to Atto647N (in red). Scale bar represents 10 µm. (c) Line profiles of the white lines displayed in (B) are shown for all three channels, each channel was normalized to its maximum signal intensity on the picture. (d) The average fluorescence of full cells, black bars or at the perinuclear regions, light-grey bars in respect to their EGFP signals were calculated. For every selected region of interest, the averaged fluorescence signal of antibodies or nanobodies was calculated and normalized to the average EGFP-fluorescence in the respective region. Statistical analysis was done using unpaired t-test, error bars represent SEM, from three independent experiments analyzing a total of 37 cells for SNAP-25-EGFP and 36 cells for Stx1A-EGFP. n.s. = not significant.

Figure Legend Snippet: Figure 4. Both nanobodies reveled epitopes that are not reported by antibodies in highly crowded regions of endogenously-expressing PC12 cells or over-expressing COS-7 cells. (a) PC12 cells were co-stained with conventional monoclonal antibodies and with our fluorescently labeled nanobodies. Monoclonal anti SNAP-25 and Syntaxin 1A (clone 71.1 and HPC-1) were detected with a secondary antibody conjugated to Abberior-Star580 (in green). S25-Nb10 or Stx1A-Nb6 were conjugated to a single Atto647N fluorophore (in red). Laser scanning confocal images of the nanobody and the antibody signals colocalize relatively good at the plasma membrane, but the nanobodies also reveal stronger signals in the perinuclear areas (especially evident for SNAP-25). The scale bar represents 10 µm. (b) Laser scanning confocal images of COS-7 cells transiently transfected with SNAP-25 or Syntaxin 1A fused to EGFP (in green) and co-stained with monoclonal primary and Cy3-fluorescently labeled secondary antibody (clone 71.1 and clone 78.2, in magenta) and nanobodies directly conjugated to Atto647N (in red). Scale bar represents 10 µm. (c) Line profiles of the white lines displayed in (B) are shown for all three channels, each channel was normalized to its maximum signal intensity on the picture. (d) The average fluorescence of full cells, black bars or at the perinuclear regions, light-grey bars in respect to their EGFP signals were calculated. For every selected region of interest, the averaged fluorescence signal of antibodies or nanobodies was calculated and normalized to the average EGFP-fluorescence in the respective region. Statistical analysis was done using unpaired t-test, error bars represent SEM, from three independent experiments analyzing a total of 37 cells for SNAP-25-EGFP and 36 cells for Stx1A-EGFP. n.s. = not significant.

Techniques Used: Expressing, Staining, Bioprocessing, Labeling, Clinical Proteomics, Membrane, Transfection, Fluorescence

Figure 6. Co-localization between SNAP-25 and Syntaxin 1A, detected by antibodies or nanobodies in STED microscopy. (a) Co-staining for SNAP-25 and Syntaxin 1A in primary hippocampal neurons. A polyclonal rabbit antibody against SNAP-25 and a mouse monoclonal against Syntaxin 1A were used. Rabbit anti SNAP-25 was further detected with a secondary antibody conjugated to Abberior-Star580; Syntaxin 1A primary antibody was detected with an Atto647N-conjugated secondary antibody. Nanobody co-staining was performed with S25-Nb10 conjugated to Abberior-Star580 and with Stx1A-Nb6 conjugated to Atto647N. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. (b) Pearson´s correlation coefficients (expressed as coefficients of determination, R2) between the green and red signals within synapses were calculated from 17 independent experiments for the antibodies and from 9 independent experiments for the nanobodies (typically, 10 images per experiment were analyzed). The values are shown as box plots, with the median, 25th and 95th percentile shown in the graph, and with symbols showing outliers. The difference is significant (Wilcoxon rank sum test; p = 0.0311).

Figure Legend Snippet: Figure 6. Co-localization between SNAP-25 and Syntaxin 1A, detected by antibodies or nanobodies in STED microscopy. (a) Co-staining for SNAP-25 and Syntaxin 1A in primary hippocampal neurons. A polyclonal rabbit antibody against SNAP-25 and a mouse monoclonal against Syntaxin 1A were used. Rabbit anti SNAP-25 was further detected with a secondary antibody conjugated to Abberior-Star580; Syntaxin 1A primary antibody was detected with an Atto647N-conjugated secondary antibody. Nanobody co-staining was performed with S25-Nb10 conjugated to Abberior-Star580 and with Stx1A-Nb6 conjugated to Atto647N. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. (b) Pearson´s correlation coefficients (expressed as coefficients of determination, R2) between the green and red signals within synapses were calculated from 17 independent experiments for the antibodies and from 9 independent experiments for the nanobodies (typically, 10 images per experiment were analyzed). The values are shown as box plots, with the median, 25th and 95th percentile shown in the graph, and with symbols showing outliers. The difference is significant (Wilcoxon rank sum test; p = 0.0311).

Techniques Used: Microscopy, Staining

Figure 8. Investigation of the extra-synaptic populations of SNAP-25 and Syntaxin 1A in hippocampal neurons upon electrical stimulation. Cultured neurons were co- stained with nanobodies directly coupled to Atto647N and with anti-Synaptophysin antibodies (detected with a secondary coupled to Alexa488) to identify synaptic regions. (a) STED images of the nanobody immunostainings were analyzed. The distribution of extra-synaptic SNAP-25 remains unchanged upon stimulation at 20 Hertz for 60 seconds, resembling the pattern previously observed in Figure 5. For control, stimulation and recovery conditions, 356, 388 and 424 synapse line profiles were analyzed from three independent experiments. (b) Upon stimulation, the population of Syntaxin 1A that is outside of synapses relocates into neighboring synapses. This population recovers if the neurons are further incubated for five minutes at 37 °C after stimulation. For control, stimulation and recovery conditions, 288, 144 and 280 synapses were analyzed from three independent experiments. The exemplary images show the nanobody signals in regions centered on synapses (determined by Synaptophysin staining). The scale bars represent 1 µm.

Figure Legend Snippet: Figure 8. Investigation of the extra-synaptic populations of SNAP-25 and Syntaxin 1A in hippocampal neurons upon electrical stimulation. Cultured neurons were co- stained with nanobodies directly coupled to Atto647N and with anti-Synaptophysin antibodies (detected with a secondary coupled to Alexa488) to identify synaptic regions. (a) STED images of the nanobody immunostainings were analyzed. The distribution of extra-synaptic SNAP-25 remains unchanged upon stimulation at 20 Hertz for 60 seconds, resembling the pattern previously observed in Figure 5. For control, stimulation and recovery conditions, 356, 388 and 424 synapse line profiles were analyzed from three independent experiments. (b) Upon stimulation, the population of Syntaxin 1A that is outside of synapses relocates into neighboring synapses. This population recovers if the neurons are further incubated for five minutes at 37 °C after stimulation. For control, stimulation and recovery conditions, 288, 144 and 280 synapses were analyzed from three independent experiments. The exemplary images show the nanobody signals in regions centered on synapses (determined by Synaptophysin staining). The scale bars represent 1 µm.

Techniques Used: Cell Culture, Staining, Control, Incubation

Produced:

Article Title: Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons.
Article Snippet: For specificity and epitope mapping analysis in Figure 2, dot blots were performed as mentioned above, but detection was performed using directly conjugated nanobodies to Atto647N fluorophore and read in the Amersham Imager 600 (GE Healthcare). .. Homolog proteins of SNAP-25 and Syntaxin 1A were all purchased from OriGene, Rockville, MD, USA and truncated versions were clone and produced as explained above. ..

Image Search Results

Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_H ABC domain, run 1.

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_H ABC domain, run 1.

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques:

Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_H ABC domain, run 2.

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Molecular dynamic simulation of interaction of Orai1 C-terminus with the STX11_H ABC domain, run 2.

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques:

Measurement of SOCE in STX11-depleted Jurkat and FHLH4 patient T cells. ( A–B ) Representative Fura-2 calcium imaging assay ( A ) and quantification of independent repeats ( B ) measuring thapsigargin (TG) induced SOCE in Jurkat T cells treated with Scr (black), STX11#1 (red) or STX11#2 (blue) shRNAs. (n=30–40 cells/group, N=3). ( C–D ) Representative Fura-2 calcium imaging assay ( C ) and quantification of repeats ( D ) showing reconstitution of SOCE in STX11-depleted Jurkat T cells by ectopic expression of STX11. Scr shRNA with empty vector (EV) expression (black), STX11 shRNA with EV expression (red), STX11 shRNA with STX11 expression (green). (n=30–40 cells/group, N=3). ( E ) Sanger sequencing of FHLH4 patient DNA showing deletion of a single adenine at the 752nd position and the resulting frameshift. ( F ) Schematic showing the predicted domain distribution of the wildtype versus FHLH4 mutant STX11 protein. ( G ) Western blot on WCLs of healthy donor (HD) and FHLH4 patient PBMCs showing the relative molecular weight and abundance of the wildtype and mutant STX11 bands (red arrow). ( H ) Western blot on WCLs of HEK293 cells over-expressing wildtype or FHLH4 mutant STX11 (red arrow) and empty vector. ( I ) Fura-2 calcium imaging assay measuring anti-CD3 induced SOCE in HD (black) and FHLH4 (red) T cells. (n=30–40 cells/group). ( J ) Fura-2 calcium imaging assay measuring SOCE in HD expressing empty vector (EV) (black) and FHLH4 T cells expressing human STX11 (red), respectively. (n=30–40 cells/group). ( K–L ) Representative Fura-2 calcium imaging assay ( K ) and quantification of repeats ( L ) measuring SOCE in Jurkat T cells treated either with Scr shRNA expressing empty vector (EV) (black), STX11 shRNA expressing EV (red), STX11 shRNA expressing wildtype STX11 (green), or STX11 shRNA expressing FHLH4 mutant STX11 (blue). Figure 1—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 1—source data 2. Folder containing the original sequencing result files, shown in . Figure 1—source data 3. Folder containing labeled PDF files of the original western blots for . Figure 1—source data 4. Folder containing the original images of western blots displayed in .

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Measurement of SOCE in STX11-depleted Jurkat and FHLH4 patient T cells. ( A–B ) Representative Fura-2 calcium imaging assay ( A ) and quantification of independent repeats ( B ) measuring thapsigargin (TG) induced SOCE in Jurkat T cells treated with Scr (black), STX11#1 (red) or STX11#2 (blue) shRNAs. (n=30–40 cells/group, N=3). ( C–D ) Representative Fura-2 calcium imaging assay ( C ) and quantification of repeats ( D ) showing reconstitution of SOCE in STX11-depleted Jurkat T cells by ectopic expression of STX11. Scr shRNA with empty vector (EV) expression (black), STX11 shRNA with EV expression (red), STX11 shRNA with STX11 expression (green). (n=30–40 cells/group, N=3). ( E ) Sanger sequencing of FHLH4 patient DNA showing deletion of a single adenine at the 752nd position and the resulting frameshift. ( F ) Schematic showing the predicted domain distribution of the wildtype versus FHLH4 mutant STX11 protein. ( G ) Western blot on WCLs of healthy donor (HD) and FHLH4 patient PBMCs showing the relative molecular weight and abundance of the wildtype and mutant STX11 bands (red arrow). ( H ) Western blot on WCLs of HEK293 cells over-expressing wildtype or FHLH4 mutant STX11 (red arrow) and empty vector. ( I ) Fura-2 calcium imaging assay measuring anti-CD3 induced SOCE in HD (black) and FHLH4 (red) T cells. (n=30–40 cells/group). ( J ) Fura-2 calcium imaging assay measuring SOCE in HD expressing empty vector (EV) (black) and FHLH4 T cells expressing human STX11 (red), respectively. (n=30–40 cells/group). ( K–L ) Representative Fura-2 calcium imaging assay ( K ) and quantification of repeats ( L ) measuring SOCE in Jurkat T cells treated either with Scr shRNA expressing empty vector (EV) (black), STX11 shRNA expressing EV (red), STX11 shRNA expressing wildtype STX11 (green), or STX11 shRNA expressing FHLH4 mutant STX11 (blue). Figure 1—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 1—source data 2. Folder containing the original sequencing result files, shown in . Figure 1—source data 3. Folder containing labeled PDF files of the original western blots for . Figure 1—source data 4. Folder containing the original images of western blots displayed in .

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques: Imaging, Expressing, shRNA, Plasmid Preparation, DNA Sequencing, Mutagenesis, Western Blot, Molecular Weight, Sequencing, Labeling

Analysis of STX11 and Orai1 mutants. ( A ) Schematic showing STX11 mutations (Top). STX11 N147A_E150A mutant expressing HEK293 co-labeled with Alexa Fluor-488 conjugated wheat germ agglutinin (WGA) or co-transfected with Orai1-YFP and immunolabeled with anti-STX11 to assess co-expression in PM of resting and store-depleted cells (Bottom). Scale bar 10 μm. ( B ) Western blot to compare total expression of mutant and wildtype STX11 using anti-STX11 antibody. ( C–D ) Representative Fura-2 calcium imaging assay ( C ) and quantification of repeats ( D ) measuring rescue of SOCE in Jurkat cells treated either with Scr shRNA (black) or STX11 shRNA expressing empty vector (red), wildtype STX11 (green), or N147A_E150A mutant STX11 (blue). ( E ) Coomassie blue stained SDS-PAGE showing expression and purification of His-tagged wildtype or N147A_E150A mutant STX11 (red arrow) from E. coli . ( F–G ) Pull-down assay measuring their binding to MBP-tagged Orai1 C-terminus. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4 ( G ) Quantification of STX11 band intensities in ( F ). ( H ) Schematic showing Orai1 mutations (Top). Confocal images of HEK293 cells expressing YFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 (Bottom). ( I–J ) Representative Fura-2 calcium imaging assay ( I ) and quantification of repeats ( J ) measuring rescue of SOCE in HEK293 cells treated either with Scr (black) or Orai1 shRNA expressing empty vector (red), wildtype Orai1 (green), or R289A_E272A_E275A_E278A mutant Orai1 (blue). ( K–L ) Pull-down assay measuring the binding of MBP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 C-terminus to wildtype or N147A_E150A mutant STX11. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4. ( L ) Quantification of STX11 band intensities in ( K ). ( M–N ) Schematic showing CAD domain in Stim1 (top panel) and representative confocal images (lower panel) ( M ) and quantification ( N ) of PM localized YFP-CAD in CFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 expressing HEK293 cells. ( O–P ) Representative Fura-2 calcium imaging assay ( O ) and quantification of repeats ( P ) of constitutive calcium flux from cells imaged in panels ( M–N ). Figure 6—source data 1. Folder containing labeled PDF files of the original gels and western blots for . Figure 6—source data 2. Folder containing the original images of gels and western blots shown in . Figure 6—source data 3. Numerical data underlying the quantitative analyses represented in .

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Analysis of STX11 and Orai1 mutants. ( A ) Schematic showing STX11 mutations (Top). STX11 N147A_E150A mutant expressing HEK293 co-labeled with Alexa Fluor-488 conjugated wheat germ agglutinin (WGA) or co-transfected with Orai1-YFP and immunolabeled with anti-STX11 to assess co-expression in PM of resting and store-depleted cells (Bottom). Scale bar 10 μm. ( B ) Western blot to compare total expression of mutant and wildtype STX11 using anti-STX11 antibody. ( C–D ) Representative Fura-2 calcium imaging assay ( C ) and quantification of repeats ( D ) measuring rescue of SOCE in Jurkat cells treated either with Scr shRNA (black) or STX11 shRNA expressing empty vector (red), wildtype STX11 (green), or N147A_E150A mutant STX11 (blue). ( E ) Coomassie blue stained SDS-PAGE showing expression and purification of His-tagged wildtype or N147A_E150A mutant STX11 (red arrow) from E. coli . ( F–G ) Pull-down assay measuring their binding to MBP-tagged Orai1 C-terminus. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4 ( G ) Quantification of STX11 band intensities in ( F ). ( H ) Schematic showing Orai1 mutations (Top). Confocal images of HEK293 cells expressing YFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 (Bottom). ( I–J ) Representative Fura-2 calcium imaging assay ( I ) and quantification of repeats ( J ) measuring rescue of SOCE in HEK293 cells treated either with Scr (black) or Orai1 shRNA expressing empty vector (red), wildtype Orai1 (green), or R289A_E272A_E275A_E278A mutant Orai1 (blue). ( K–L ) Pull-down assay measuring the binding of MBP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 C-terminus to wildtype or N147A_E150A mutant STX11. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4. ( L ) Quantification of STX11 band intensities in ( K ). ( M–N ) Schematic showing CAD domain in Stim1 (top panel) and representative confocal images (lower panel) ( M ) and quantification ( N ) of PM localized YFP-CAD in CFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 expressing HEK293 cells. ( O–P ) Representative Fura-2 calcium imaging assay ( O ) and quantification of repeats ( P ) of constitutive calcium flux from cells imaged in panels ( M–N ). Figure 6—source data 1. Folder containing labeled PDF files of the original gels and western blots for . Figure 6—source data 2. Folder containing the original images of gels and western blots shown in . Figure 6—source data 3. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques: Analysis, Mutagenesis, Expressing, Labeling, Transfection, Immunolabeling, Western Blot, Imaging, shRNA, Plasmid Preparation, Staining, SDS Page, Purification, Pull Down Assay, Binding Assay, Confocal

Intracellular localization of STX11 and its association with Orai1. ( A ) (Upper panel) Schematic showing epitopes for anti-STX11 and anti-HA antibodies used in the lower panel. (Lower panel) Representative confocal images of HEK293 cells transfected with either HA-tagged STX11 or untagged STX11 and stained using anti-HA antibody or anti-STX11 antibody, respectively. N=3 ( B–D ) Localization of HA-tagged STX11 with respect to Orai1-YFP in the basal as well as middle plane of resting ( B ) and store-depleted ( C ) HEK293 cells. ( D ) Pearson’s correlation coefficient showing Orai1 and STX11 colocalization from ( B ) and ( C ). N=3. ( E ) Representative confocal images of FHLH4 mutant STX11 expressing HEK293 cells. ( F–G ) Co-IP to assess STX11 binding to Orai1. Whole cell lysates of resting and store-depleted HEK293 cells co-expressing either Flag-Orai1 and STX11 ( F ) or Orai1-Myc and STX11 ( G ) were subjected to IP and western blot using anti-Myc, anti-Flag, or anti-STX11 antibodies, as indicated. (N=3) ( H ) Schematic showing key domains of STX11 and Orai1 used for in vitro pull-down assays. ( I ) Pull-down assay showing in vitro binding of His-tagged STX11 to Orai1. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. Input: 1/5 th of the protein. N=4. ( J ) Pull-down assay showing in vitro binding of His-tagged H abc domain of STX11 to Orai1 fragments. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-His antibody. Input: 1/5 th of the protein. N=3. ( K ) Binding free energy distribution of STX11-H abc and Orai1 C-terminus interactions. ( L ) Sphere and cartoon representation of the structure of STX11 H abc (green) and Orai1 C-terminus (cyan) complex after MD simulation. The N-termini are highlighted in blue, and C-termini in red. ( M ) Representative confocal images showing localization of STX11 with respect to Orai1:Stim1 puncta in store-depleted cells. HEK293 cells expressing Orai1-YFP, CFP-Stim1 were transfected with HA-tagged STX11, store-depleted, and stained using anti-HA antibody. (N=3) ( N ) Pearson’s correlation coefficient showing quantification of the localization of STX11 with respect to Stim1:Orai1 puncta in store-depleted cells from ( M ). Figure 5—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 5—source data 2. Folder containing labeled PDF files of the original western blots for indicating the relevant bands. Figure 5—source data 3. Folder containing the original images of western blots shown in .

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Intracellular localization of STX11 and its association with Orai1. ( A ) (Upper panel) Schematic showing epitopes for anti-STX11 and anti-HA antibodies used in the lower panel. (Lower panel) Representative confocal images of HEK293 cells transfected with either HA-tagged STX11 or untagged STX11 and stained using anti-HA antibody or anti-STX11 antibody, respectively. N=3 ( B–D ) Localization of HA-tagged STX11 with respect to Orai1-YFP in the basal as well as middle plane of resting ( B ) and store-depleted ( C ) HEK293 cells. ( D ) Pearson’s correlation coefficient showing Orai1 and STX11 colocalization from ( B ) and ( C ). N=3. ( E ) Representative confocal images of FHLH4 mutant STX11 expressing HEK293 cells. ( F–G ) Co-IP to assess STX11 binding to Orai1. Whole cell lysates of resting and store-depleted HEK293 cells co-expressing either Flag-Orai1 and STX11 ( F ) or Orai1-Myc and STX11 ( G ) were subjected to IP and western blot using anti-Myc, anti-Flag, or anti-STX11 antibodies, as indicated. (N=3) ( H ) Schematic showing key domains of STX11 and Orai1 used for in vitro pull-down assays. ( I ) Pull-down assay showing in vitro binding of His-tagged STX11 to Orai1. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. Input: 1/5 th of the protein. N=4. ( J ) Pull-down assay showing in vitro binding of His-tagged H abc domain of STX11 to Orai1 fragments. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-His antibody. Input: 1/5 th of the protein. N=3. ( K ) Binding free energy distribution of STX11-H abc and Orai1 C-terminus interactions. ( L ) Sphere and cartoon representation of the structure of STX11 H abc (green) and Orai1 C-terminus (cyan) complex after MD simulation. The N-termini are highlighted in blue, and C-termini in red. ( M ) Representative confocal images showing localization of STX11 with respect to Orai1:Stim1 puncta in store-depleted cells. HEK293 cells expressing Orai1-YFP, CFP-Stim1 were transfected with HA-tagged STX11, store-depleted, and stained using anti-HA antibody. (N=3) ( N ) Pearson’s correlation coefficient showing quantification of the localization of STX11 with respect to Stim1:Orai1 puncta in store-depleted cells from ( M ). Figure 5—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 5—source data 2. Folder containing labeled PDF files of the original western blots for indicating the relevant bands. Figure 5—source data 3. Folder containing the original images of western blots shown in .

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques: Confocal, Transfection, Staining, Mutagenesis, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, In Vitro, Pull Down Assay, Labeling

$( A ) Co-immunoprecipitation (co-IP) to test the association of Stim1 with STX11. HEK293 cells were co-transfected with Stim1-Myc-His and STX11, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2) ( B ) Quantification of the fraction of Orai1 inside and outside Stim1:Orai1 clusters in store-depleted HEK293 cells expressing CFP-Orai1 and Stim1-YFP. Figure 5—figure supplement 4—source data 1. Folder containing labeled PDF files of the original western blots for . Figure 5—figure supplement 4—source data 2. Folder containing the original images of western blots shown in . Figure 5—figure supplement 4—source data 3. Numerical data underlying the quantitative analyses represented in .$

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (co-IP) to test the association of Stim1 with STX11. HEK293 cells were co-transfected with Stim1-Myc-His and STX11, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2) ( B ) Quantification of the fraction of Orai1 inside and outside Stim1:Orai1 clusters in store-depleted HEK293 cells expressing CFP-Orai1 and Stim1-YFP. Figure 5—figure supplement 4—source data 1. Folder containing labeled PDF files of the original western blots for . Figure 5—figure supplement 4—source data 2. Folder containing the original images of western blots shown in . Figure 5—figure supplement 4—source data 3. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, Labeling

( A ) Schematic showing a comparison of the key domains in STX11 and STX1A. ( B ) Western blot of whole cell lysates prepared from HEK293 cells expressing YFP-STX11 and STX11-YFP. Note the mismatch in molecular weight of the N- versus C-terminally tagged STX11 and the degradation products (arrows). N=3. ( C ) Confocal images showing localization of ectopically expressed mCherry-STX11 or STX11-mCherry in HEK293 cells. N=3. ( D ) Pull-down assay showing in vitro binding of His-tagged SNARE domain of STX11 to MBP-tagged Orai1 N- and C-termini. (Top panel) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 cytosolic tails. (Bottom panel) Western blot using anti-His antibody. Figure 5—figure supplement 1—source data 1. Folder containing labeled PDF files of the original western blots for . Figure 5—figure supplement 1—source data 2. Folder containing the original images of western blots shown in .

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Schematic showing a comparison of the key domains in STX11 and STX1A. ( B ) Western blot of whole cell lysates prepared from HEK293 cells expressing YFP-STX11 and STX11-YFP. Note the mismatch in molecular weight of the N- versus C-terminally tagged STX11 and the degradation products (arrows). N=3. ( C ) Confocal images showing localization of ectopically expressed mCherry-STX11 or STX11-mCherry in HEK293 cells. N=3. ( D ) Pull-down assay showing in vitro binding of His-tagged SNARE domain of STX11 to MBP-tagged Orai1 N- and C-termini. (Top panel) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 cytosolic tails. (Bottom panel) Western blot using anti-His antibody. Figure 5—figure supplement 1—source data 1. Folder containing labeled PDF files of the original western blots for . Figure 5—figure supplement 1—source data 2. Folder containing the original images of western blots shown in .

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques: Comparison, Western Blot, Expressing, Molecular Weight, Confocal, Pull Down Assay, In Vitro, Binding Assay, Staining, Labeling

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-STX11 , Proteintech , 13301–1-AP (RRID: AB_2198214 ) , IF (0.3 μg/ml).

Techniques:

(A) Self-quenching of Alexa 647 attached to the C-terminus of Syntaxin was used to probe oligomerization of Syntaxin in proteoliposomes. (B) Alexa 647 fluorescence intensity normalized to bPC:Chol Syx condition. Kruskal-Wallis test for non-Gaussian distribution was used for statistical analysis. (C) Probing the state of Q-SNAREs by solubilizing Q-SNARE proteoliposomes in SDS. (D) SDS gel electrophoresis of solubilized Q-SNAREs and Western blotting with anti-Syntaxin-1a (left) and anti-SNAP25 (right) antibodies. (E and F) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (E) and Syntaxin/SNAP25 (F) samples. In (F) oligomer populations are marked with asterisks to indicate that they might consist of the mixture of Syntaxin’s homo-oligomers and Syntaxin/SNAP25 hetero-oligomers. Two-way ANOVA was used. Error bars represent standard error.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Self-quenching of Alexa 647 attached to the C-terminus of Syntaxin was used to probe oligomerization of Syntaxin in proteoliposomes. (B) Alexa 647 fluorescence intensity normalized to bPC:Chol Syx condition. Kruskal-Wallis test for non-Gaussian distribution was used for statistical analysis. (C) Probing the state of Q-SNAREs by solubilizing Q-SNARE proteoliposomes in SDS. (D) SDS gel electrophoresis of solubilized Q-SNAREs and Western blotting with anti-Syntaxin-1a (left) and anti-SNAP25 (right) antibodies. (E and F) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (E) and Syntaxin/SNAP25 (F) samples. In (F) oligomer populations are marked with asterisks to indicate that they might consist of the mixture of Syntaxin’s homo-oligomers and Syntaxin/SNAP25 hetero-oligomers. Two-way ANOVA was used. Error bars represent standard error.

Article Snippet: Antibodies for Syntaxin-1a (mouse monoclonal, catalog number 110 011), SNAP-25 (rabbit polyclonal, catalog number 111 002), and Munc18 (mouse monoclonal, catalog number 116 011) were from Synaptic Systems.

Techniques: Fluorescence, SDS-Gel, Electrophoresis, Western Blot

(A) TIRF microscopy was used to examine binding of fluorescently labeled Munc18 to planar supported membranes. (B) TIRF microscopy binding curves of 0.5 µM Munc18 to planar supported membranes containing Syntaxin only. (C) Maximum fluorescence intensity values obtained from fits of the binding curves from (B) to 1 st order kinetic functions. (D) TIRF microscopy binding curves of 0.5 µM Munc18 to planar supported membranes containing Syntaxin co-reconstituted with SNAP25. (E) Maximum fluorescence intensity values obtained from fits of the binding curves from (D) to 1 st order kinetic functions. Error bars represent standard error. T-test with Welch’s correction for unequal SDs was used in (C) and (E) . We additionally used t-test with Welch’s correction to compare maximum fluorescence intensity values between Syntaxin only and Syntaxin:SNAP25 membranes of each lipid condition. Differences were statistically significant, with p=0.0001 for PM 1% PIP2 membranes, and p=0.0113 for bPC:Chol membranes.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) TIRF microscopy was used to examine binding of fluorescently labeled Munc18 to planar supported membranes. (B) TIRF microscopy binding curves of 0.5 µM Munc18 to planar supported membranes containing Syntaxin only. (C) Maximum fluorescence intensity values obtained from fits of the binding curves from (B) to 1 st order kinetic functions. (D) TIRF microscopy binding curves of 0.5 µM Munc18 to planar supported membranes containing Syntaxin co-reconstituted with SNAP25. (E) Maximum fluorescence intensity values obtained from fits of the binding curves from (D) to 1 st order kinetic functions. Error bars represent standard error. T-test with Welch’s correction for unequal SDs was used in (C) and (E) . We additionally used t-test with Welch’s correction to compare maximum fluorescence intensity values between Syntaxin only and Syntaxin:SNAP25 membranes of each lipid condition. Differences were statistically significant, with p=0.0001 for PM 1% PIP2 membranes, and p=0.0113 for bPC:Chol membranes.

Techniques: Microscopy, Binding Assay, Labeling, Fluorescence

(A) Design of the co-floatation assay in a Nycodenz density gradient. (B) Distribution of lipids probed by NBD-DOPE in PM proteoliposomes in the gradient. (C) Distribution of Syntaxin in PM and bPC:Chol proteoliposomes in the gradient. (D, E) Distribution of Munc18 in the gradient with PM and bPC:Chol proteoliposomes with Syntaxin only (D) and Syntaxin co-reconstituted with SNAP25 (E) . Error bars indicate standard error of the mean.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Design of the co-floatation assay in a Nycodenz density gradient. (B) Distribution of lipids probed by NBD-DOPE in PM proteoliposomes in the gradient. (C) Distribution of Syntaxin in PM and bPC:Chol proteoliposomes in the gradient. (D, E) Distribution of Munc18 in the gradient with PM and bPC:Chol proteoliposomes with Syntaxin only (D) and Syntaxin co-reconstituted with SNAP25 (E) . Error bars indicate standard error of the mean.

Techniques:

(A) Change of fluorescence intensity of Syntaxin-289C-Alexa647 in bPC:Chol membranes after the addition of 0.5 µM Munc18. (B and C) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (B) and Syntaxin/SNAP25 (C) samples in bPC:Chol membranes before and after incubation with 0.5 µM Munc18. (D) Change of fluorescence intensity of Syntaxin-289C-Alexa647 in PM membranes after the addition of 0.5 µM Munc18. (E and F) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (E) and Syntaxin/SNAP25 (F) samples in PM membranes before and after incubation with 0.5 µM Munc18.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Change of fluorescence intensity of Syntaxin-289C-Alexa647 in bPC:Chol membranes after the addition of 0.5 µM Munc18. (B and C) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (B) and Syntaxin/SNAP25 (C) samples in bPC:Chol membranes before and after incubation with 0.5 µM Munc18. (D) Change of fluorescence intensity of Syntaxin-289C-Alexa647 in PM membranes after the addition of 0.5 µM Munc18. (E and F) Quantification of populations of oligomeric states of Syntaxin and SNAP25 from Syntaxin only (E) and Syntaxin/SNAP25 (F) samples in PM membranes before and after incubation with 0.5 µM Munc18.

Techniques: Fluorescence, Incubation

(A) Visual representation of residues of Syntaxin that were site-specifically labeled with Alexa 546 for FLIC microscopy experiments. The N-peptide is marked green, the H abc domain is marked orange, the SNARE motif is marked red, and the 7 labeled residues are indicated. (B, C) Distances of the labeled sites of Syntaxin from the membrane before (solid bars) and after (open bars) incubation with 0.5 µM Munc18 in PM 1% PIP2 supported lipid bilayers with Syntaxin only (B) or Syntaxin co-reconstituted with SNAP25 (C) . (D) Distance of residue 192 of Syntaxin (N-terminal end of SNARE motif) co-reconstituted with SNAP25 in bPC:Chol and PM 1% PIP2 lipid bilayers before and after incubation with 0.4 µM Munc18. Error bars represent standard error. For statistical analysis, one-way ANOVA test was used, asterisks indicate ** p=0.003 and **** p<0.0001.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Visual representation of residues of Syntaxin that were site-specifically labeled with Alexa 546 for FLIC microscopy experiments. The N-peptide is marked green, the H abc domain is marked orange, the SNARE motif is marked red, and the 7 labeled residues are indicated. (B, C) Distances of the labeled sites of Syntaxin from the membrane before (solid bars) and after (open bars) incubation with 0.5 µM Munc18 in PM 1% PIP2 supported lipid bilayers with Syntaxin only (B) or Syntaxin co-reconstituted with SNAP25 (C) . (D) Distance of residue 192 of Syntaxin (N-terminal end of SNARE motif) co-reconstituted with SNAP25 in bPC:Chol and PM 1% PIP2 lipid bilayers before and after incubation with 0.4 µM Munc18. Error bars represent standard error. For statistical analysis, one-way ANOVA test was used, asterisks indicate ** p=0.003 and **** p<0.0001.

Techniques: Labeling, Microscopy, Membrane, Incubation, Residue

(A) Docking and fusion of insulin granules containing C-peptide-GFP with Q-SNARE-containing planar supported membranes were imaged using TIRF microscopy. (B) Example of a fluorescence intensity trace with marked onsets of docking and fusion, and the docking to fusion delay time dt. (C) Docking of granules to supported PM 1% PIP2 or bPC:Chol Syntaxin/SNAP25 membranes without and with Munc18, normalized to the number of docked granules with PM 1% PIP2 membranes without Munc18. (D) Fusion efficiency measured as a percentage of docked granules that proceeded to fusion under the same conditions as in (C) . (E-J) Analysis of fusion kinetics. (E and F) Comparison of docking to fusion delay times with and without the addition of Munc18 in PM 1% PIP2 membranes (E) and bPC:Chol membranes (F) . (G and H) Cumulative distribution function of the delay times (data points) and the fit with a parallel reaction model N ( t ) = (1 − exp − kt ) m , where k is the rate constant and m is the number of parallel reactions occurring (solid line) in PM 1% PIP2 (G) and bPC:Chol (H) membranes. (I and J) Half-times of the maximum fusion calculated from the fits of the cumulative distribution function of the delay times in PM 1% PIP2 (I) and bPC:Chol membranes (J) . For statistical analysis, Kruskal-Wallis test for non-Gaussian distribution was used in multiple comparison tests. Kolmogorov-Smirnov test for cumulative distributions was used to compare docking to fusion delay times.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Docking and fusion of insulin granules containing C-peptide-GFP with Q-SNARE-containing planar supported membranes were imaged using TIRF microscopy. (B) Example of a fluorescence intensity trace with marked onsets of docking and fusion, and the docking to fusion delay time dt. (C) Docking of granules to supported PM 1% PIP2 or bPC:Chol Syntaxin/SNAP25 membranes without and with Munc18, normalized to the number of docked granules with PM 1% PIP2 membranes without Munc18. (D) Fusion efficiency measured as a percentage of docked granules that proceeded to fusion under the same conditions as in (C) . (E-J) Analysis of fusion kinetics. (E and F) Comparison of docking to fusion delay times with and without the addition of Munc18 in PM 1% PIP2 membranes (E) and bPC:Chol membranes (F) . (G and H) Cumulative distribution function of the delay times (data points) and the fit with a parallel reaction model N ( t ) = (1 − exp − kt ) m , where k is the rate constant and m is the number of parallel reactions occurring (solid line) in PM 1% PIP2 (G) and bPC:Chol (H) membranes. (I and J) Half-times of the maximum fusion calculated from the fits of the cumulative distribution function of the delay times in PM 1% PIP2 (I) and bPC:Chol membranes (J) . For statistical analysis, Kruskal-Wallis test for non-Gaussian distribution was used in multiple comparison tests. Kolmogorov-Smirnov test for cumulative distributions was used to compare docking to fusion delay times.

Techniques: Microscopy, Fluorescence, Comparison

(A) Cryo-EM micrographs of PM 1% PIP2 proteoliposomes reconstituted with Munc18/Syntaxin/SNAP25. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (B) 2D class averages of Munc18/Syntaxin/SNAP25 in proteoliposomes. The scale bar is 120 Å. (C) Cryo-EM reconstruction of Munc18/Syntaxin/SNAP25 complex in proteoliposomes. (D) Cryo-EM micrographs of nanodiscs containing Munc18/Syntaxin complexes. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (E) 2D class averages of Munc18/Syntaxin in nanodiscs. The scale bar is 110 Å. (F) Cryo-EM reconstruction of protein complex from Munc18/Syntaxin in nanodiscs. (G) Cryo-EM micrographs of nanodiscs containing Munc18/Syntaxin/SNAP25 complexes. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (H) 2D class averages of Munc18/Syntaxin/SNAP25 in nanodiscs. The scale bar is 110 Å. (I) Cryo-EM reconstruction of protein complex from Munc18/Syntaxin/SNAP25 in nanodiscs.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Cryo-EM micrographs of PM 1% PIP2 proteoliposomes reconstituted with Munc18/Syntaxin/SNAP25. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (B) 2D class averages of Munc18/Syntaxin/SNAP25 in proteoliposomes. The scale bar is 120 Å. (C) Cryo-EM reconstruction of Munc18/Syntaxin/SNAP25 complex in proteoliposomes. (D) Cryo-EM micrographs of nanodiscs containing Munc18/Syntaxin complexes. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (E) 2D class averages of Munc18/Syntaxin in nanodiscs. The scale bar is 110 Å. (F) Cryo-EM reconstruction of protein complex from Munc18/Syntaxin in nanodiscs. (G) Cryo-EM micrographs of nanodiscs containing Munc18/Syntaxin/SNAP25 complexes. Scale bars for the images are approximately 200 Å (left) and 100 Å (right). (H) 2D class averages of Munc18/Syntaxin/SNAP25 in nanodiscs. The scale bar is 110 Å. (I) Cryo-EM reconstruction of protein complex from Munc18/Syntaxin/SNAP25 in nanodiscs.

Techniques: Cryo-EM Sample Prep

(A) Side-by-side comparisons of the two density maps when opened together in ChimeraX. (B) Comparison of the protein density in the two maps when they are fit into each other in ChimeraX. (C) Additional views of the comparison of the two maps when they are fit into each other in ChimeraX. The black and pink arrows point to the main and secondary extra densities, respectively, that are observed in Munc18/Syntaxin/SNAP25 but lacking in Munc18/Syntaxin.

Journal: bioRxiv

Article Title: Munc18 binds to and organizes membrane-bound acceptor Q-SNARE complexes in a fashion that depends on the membrane’s lipid composition

doi: 10.64898/2026.07.14.738512

Figure Lengend Snippet: (A) Side-by-side comparisons of the two density maps when opened together in ChimeraX. (B) Comparison of the protein density in the two maps when they are fit into each other in ChimeraX. (C) Additional views of the comparison of the two maps when they are fit into each other in ChimeraX. The black and pink arrows point to the main and secondary extra densities, respectively, that are observed in Munc18/Syntaxin/SNAP25 but lacking in Munc18/Syntaxin.

Techniques: Comparison

Review

syntaxin 1a (OriGene)

Structured Review

Images

1) Product Images from "Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons."

Related Articles

Produced:

Similar Products

Image Search Results