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:

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:

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

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

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

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

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:

Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation drives plasma membrane accumulation of NS3/NS3A. ( A ) The stability of NS3/NS3A WT and NS3/NS3A N150Q proteins was examined in both transfected (top panels) and BTV-20-infected cells (bottom panels; MOI = 10). For transfection assays, HEK-293T cells were transfected with plasmids expressing NS3/NS3A WT or the N150Q mutant and treated with cycloheximide (CHX; 100 μg/mL) at 18 h post-transfection (designated as 0 h post-CHX treatment) to block de novo protein synthesis. For infection assays, MDOK cells were infected with BTV-20 WT or BTV-20 N150Q and treated with CHX (100 μg/mL) at 10 h post-infection (designated as 0 h post-CHX treatment). Cells were harvested at the indicated time points and analyzed by Western blotting. ( B ) Quantification of NS3/NS3A protein levels shown in panel A was performed by ImageJ densitometric analysis. Protein levels at each time point were normalized to the corresponding 0 h post-CHX treatment (18 h post-transfection or 10 h post-infection, respectively). ( C ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with ER marker anti-calnexin (green) or Golgi marker anti-syntaxin 6 (green). Fluorescence distribution was evaluated using line-scan intensity profiles. Scale bar, 5 µm. ( D ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with plasma membrane marker WGA-Alexa Fluor 488 (green). Line-scan intensity profiles are shown. Scale bar, 5 µm. ( E ) Plasma membrane isolation of HEK-293T cells transfected with NS3/NS3A WT or N150Q mutant, followed by Western blot analysis. PM (plasma membrane fraction); NPM (non-plasma membrane fraction); Total (plasma membrane fraction + non-plasma membrane fraction). ( F ) Quantification of NS3/NS3A at the plasma membrane fraction was analyzed by Image J from panel E (* P < 0.05, two-tailed unpaired t-test). ( G ) Confocal imaging of HeLa cells co-transfected with NS3/NS3A (WT or N150Q, red) and VP2 (green) or VP5 (green), showing their subcellular colocalization. Colocalization was assessed by line-scan intensity profiles.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Transfection, Infection, Expressing, Mutagenesis, Blocking Assay, Western Blot, Staining, Marker, Fluorescence, Isolation, Two Tailed Test, Imaging