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–C ) Estimation of nuclear translocation of NFAT. ( A ) Western blot showing nuclear translocation of NFAT in Jurkat T cells treated with Scr or STX11 shRNA and stimulated with PMA +TG. N=3. ( B ) Representative confocal images of control and STX11-depleted Jurkat T cells stimulated with anti-CD3, immunolabeled with anti-NFAT1 antibody, and counter-stained with DAPI. (N=3) ( C ) Box and whisker plot showing quantification of nuclear NFAT from cells populating 10 randomly chosen fields per group in ( B ). ( D ) Quantification of IL-2 mRNA in anti-CD3 stimulated Jurkat T cells treated with Scr or STX11 shRNA using qPCR. The bars show mean ± SE of relative IL-2 mRNA. N=3. ( E ) Quantification of IL-2 EIA on supernatants of Scr (black) or STX11 (red) shRNA-treated Jurkat T cells stimulated with anti-CD3. ( F ) qPCR analysis of IL-2 mRNA expression in various stimulated HD (black) and FHLH4 T cells (red). ( G–H ) Flow cytometry contour plot showing granule release assay performed on HD (black) and FHLH4 CD8 T (red) cells ( G ) and its quantification ( H ). Figure 4—source data 1. Folder containing labeled PDF files of original western blots for . Figure 4—source data 2. Folder containing the original images of western blots shown in . Figure 4—source data 3. Numerical data underlying the quantitative analyses represented in . Figure 4—source data 4. Folder containing the original flow cytometry files and gating strategy for Figure 4G .

Article Snippet: Other , 30% Acrylamide:Bis Solution (29:1) , Biorad , 1610156 , SDS-PAGE gels in , — and and their associated figure supplements .

Techniques: Translocation Assay, Western Blot, shRNA, Confocal, Control, Immunolabeling, Staining, Whisker Assay, Analysis, Expressing, Flow Cytometry, Release 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: 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: Other , 30% Acrylamide:Bis Solution (29:1) , Biorad , 1610156 , SDS-PAGE gels in , — and and their associated figure supplements .

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: Other , 30% Acrylamide:Bis Solution (29:1) , Biorad , 1610156 , SDS-PAGE gels in , — and and their associated figure supplements .

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: Oligomerization of Orai1. ( A ) N-FRET analysis of Scr and STX11 shRNA-treated HEK293 cells co-expressing Orai1-CFP and Orai1-YFP before and after store-depletion. ( B–C ) Representative images ( B ) and quantification ( C ) of FRET analysis using acceptor photobleaching of Scr and STX11 shRNA-treated Orai1, Stim1 double knockdown resting HEK293 cells co-expressing Orai1-CFP (donor) and Orai1-YFP (acceptor). ( D–E ) Representative western blot ( D ) and quantification of band intensities from repeats ( E ) showing distribution of BS3 crosslinked Flag-Orai1 oligomeric bands B,C,D in control and STX11-depleted HEK293 cells. N=3. ( F ) Representative confocal images and quantification (right) of YFP-CAD localization in Scr and STX11 shRNA-treated HEK293 cells expressing Orai1-CFP. ( G–H ) Fura-2 calcium imaging assay ( G ) and quantification of repeats ( H ) to measure constitutive calcium influx in Orai1-CFP and YFP-CAD expressing, control (black) or STX11-depleted (red) HEK293 cells. n=80–90, N=3 ( I–J ) Fura-2 calcium imaging assay ( I ) and quantification of repeats ( J ) to measure constitutive calcium influx in Orai1-H134S mutant expressing control (black) and STX11-depleted (red) HEK293 cells. n=80–100, N=3 ( K–L ) Fura-2 calcium imaging assay ( K ) and quantification of repeats ( L ) to measure constitutive calcium influx in Orai1-ANSGA mutant expressing control (black) and STX11-depleted (red) HEK293 cells. n=50–60, N=3. Figure 8—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 8—source data 2. Folder containing labeled PDF files of the original western blots for . Figure 8—source data 3. Folder containing the original images of western blots shown in .

Article Snippet: Other , 30% Acrylamide:Bis Solution (29:1) , Biorad , 1610156 , SDS-PAGE gels in , — and and their associated figure supplements .

Techniques: Analysis, shRNA, Expressing, Knockdown, Western Blot, Control, Confocal, Imaging, Mutagenesis, Labeling, Förster Resonance Energy Transfer

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: Other , 30% Acrylamide:Bis Solution (29:1) , Biorad , 1610156 , SDS-PAGE gels in , — and and their associated figure supplements .

Techniques: