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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 ,
Techniques:
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 ,
Techniques:
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 ,
Techniques: Imaging, Expressing, shRNA, Plasmid Preparation, DNA Sequencing, Mutagenesis, Western Blot, Molecular Weight, Sequencing, 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: 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 ,
Techniques: Analysis, Mutagenesis, Expressing, Labeling, Transfection, Immunolabeling, Western Blot, Imaging, shRNA, Plasmid Preparation, Staining, SDS Page, Purification, Pull Down Assay, Binding Assay, Confocal
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 ,
Techniques: Confocal, Transfection, Staining, Mutagenesis, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, In Vitro, Pull Down Assay, 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: ( 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 ,
Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, 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: ( 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 ,
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 ,
Techniques:
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
Techniques: Fluorescence, SDS-Gel, Electrophoresis, Western Blot
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.
Article Snippet: Antibodies for
Techniques: Microscopy, Binding Assay, Labeling, Fluorescence
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.
Article Snippet: Antibodies for
Techniques:
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.
Article Snippet: Antibodies for
Techniques: Fluorescence, Incubation
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.
Article Snippet: Antibodies for
Techniques: Labeling, Microscopy, Membrane, Incubation, Residue
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.
Article Snippet: Antibodies for
Techniques: Microscopy, Fluorescence, Comparison
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.
Article Snippet: Antibodies for
Techniques: Cryo-EM Sample Prep
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.
Article Snippet: Antibodies for
Techniques: Comparison