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

Journal: The Journal of Biological Chemistry

Article Title: O -Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking *

doi: 10.1074/jbc.M115.711838

Figure Lengend Snippet: Endocytic trafficking of PAM-1/OSX is altered. The endocytic trafficking of PAM-1 and PAM-1/OSX was monitored by incubating live cells with ectodomain antibody to PAM. A, cells incubated with PAM antibody for 5 min were chased for 15 min, fixed, and permeabilized; endogenous syntaxin 6 and internalized ectodomain antibody were visualized. Scale bars, 10 μm. B, cells incubated with PAM antibody-gold complexes at 4 °C were visualized after a 20-min chase at 37 °C. Representative images are shown as follows: three labeled multivesicular bodies are seen in the PAM-1 cell and one in the PAM-1/OSX cell; insets show labeled tubular structures. Scale bars, 200 nm. C, graph shows the percentage of total gold particles in tubular structures, early endosomes (EE), multivesicular bodies (MVB), and lysosomes (Lys) in PAM-1 (gray bars) and PAM-1/OSX (black bars) cells after the 20-min chase (mean ± S.E.; *, p < 0.001). D, PAM-1 and PAM-1/OSX cells exposed to PAM antibody and to fluorescently tagged WGA for 5 min were rinsed and chased for 5 or 10 min before fixation; internalized antibody was visualized after permeabilization. Scale bars, 10 μm. E and F, PAM-1 and PAM-1/OSX cells kept on ice were incubated with PAM antibody-gold complexes and WGA-HRP; cells were fixed after a chase incubation at 37 °C for 1, 2, or 5 min. E, after a 1- or 2-min chase, co-localized PAM antibody-gold complexes and peroxidase product are indicated by arrows; open arrows mark PAM-1 separated from WGA and in tubules. Scale bars, 200 nm. F, after the 5-min chase, antibody/gold particles were found in tubular structures and early endosomes (EE) in PAM-1 cells; in PAM-1/OSX cells, antibody/gold particles were confined to tubular structures localized peripherally or near the Golgi complex (G, Golgi stack; IMG, immature secretory granule). Scale bars, 200 nm.

Article Snippet: Antibody internalization studies were carried out as described ( 31 ) using a PAM ectodomain antibody (JH629 or JH471, 1:1000) ( 3 ) and Alexa Fluor 488-conjugated wheat germ agglutinin (WGA) (1 μg/ml Molecular Probes) for 5 or 10 min. After a 5–35-min chase, cells were fixed with 4% paraformaldehyde, permeabilized using 0.125% Triton X-100, and incubated with monoclonal antibody to syntaxin 6 (1:100; BD Transduction Laboratories) for 1 h at room temperature.

Techniques: Incubation, Labeling

Journal: Gene Expression

Article Title: Molecular Cloning of the m- Golsyn Gene and its Expression in the Mouse Brain

doi:

Figure Lengend Snippet: Subcellular distribution of m-Golsyn protein in mouse cerebral cortex. (A) Procedure for subcellular fractionation of mouse cerebral cortex. Homogenates were separated into fractions enriched in nuclei (N), 100,000 × g supernatant (S), 100,000 × g precipitate (P), synaptic plasma membrane (SM), presynaptic cytosol (PC), and synaptic vesicle (SV) by differential centrifugation. (B) Fractions were prepared as shown in (A), and an aliquot (20 μg protein) of each of these fractions was resolved by 7.5% SDS-PAGE and subjected to immunoblot analysis with antibodies against GOLSYN, synaptophysin, syntaxin 6, or PDI. Synaptophysin, syntaxin 6, and PDI were used as markers for synaptic vesicle, Golgi apparatus, and endoplasmic reticulum, respectively. Solid and gray arrowheads indicate the position of m-Golsyn and PDI, respectively. (C) P and SV fractions, prepared as shown in (A), were fractionated by ultracentrifugation of a sucrose gradient and subjected to immunoblot analysis.

Article Snippet: The following antibodies were purchased from the sources indicated: mouse anti-protein disulfide isomerase (PDI) antibody, from StressGen Biotechnologies (Canada, BC); mouse anti-syntaxin 6 antibody, from BD Transduction (San Diego, CA); mouse anti-synaptophysin antibody and mouse anti-β-tubulin antibody, from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mouse anti-neuronal nuclei (NeuN) monoclonal antibody, from Chemicon International, Inc. (Temecula, CA); mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody and FITC-conjugated anti-mouse IgG antibody, from Sigma Chemical Co. (St. Louis, MO); horseradish peroxidase (HRP)-conjugated, swine anti-rabbit immunoglobulins, from DakoCytomation Inc. (Car-pinteria, CA); peroxidase-linked anti-mouse Ig, from Amersham Biosciences Corp. (Piscataway, NJ); biotinylated anti-rabbit IgG antibody, from Vector Laboratories Inc. (Burlingame, CA); Texas Red-conjugated goat anti-rabbit IgG, from Molecular Probes Inc. (Eugene, OR).

Techniques: Fractionation, Centrifugation, SDS Page, Western Blot

Journal: Blood

Article Title: Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6

doi: 10.1182/blood-2010-06-291690

Figure Lengend Snippet: VEGF stimulates exit of VEGFR2 from the trans-Golgi complex. (A) Serum-starved HUVECs were labeled with mAb against VEGFR2 (55B11) and TGN46 (Golgi marker). Total cell-associated and Golgi-localized fluorescence intensity of VEGFR2 was quantified by image analysis. Values are expressed as a fraction of the total VEGFR2 in the Golgi apparatus. (B) Homogenates prepared from serum-starved HUVECs were fractionated on a self-generated Optiprep gradient (10%, 20%, 30%) and immunoblotted with antibodies against proteins enriched in the PM (vascular endothelial-Cadherin); the trans-Golgi complex (TGN46); or endosomes (EEA1). (C) Percentage of total VEGFR2 and TGN46 in each fraction, based on quantification of density of bands in each fraction obtained by Optiprep gradient centrifugation. (D) Effects of VEGF-A treatment on VEGFR2 localization at the Golgi apparatus. Serum-starved HUVECs were treated with CHX (10 μg/mL), and immunofluorescence imaging was carried out for VEGFR2 and TGN46 localization. (E) Quantification of the Golgi-localized VEGFR2 (overlapping with TGN46) shown in panel D. Values are expressed as a percentage of change in intensity of Golgi-localized VEGFR2 signal (relative to initial intensity in at 0 minutes chase at 37°C, data not shown). Percentages in panels A and E represent mean (± SD) in n = 90 cells for each condition from 5 separate experiments. For panel E, P ≤ .05. (F-G) Effects of BFA treatment on VEGFR2 transport in HUVECs. (F) Representative images of immunofluorescence analysis of untreated and BFA-treated cells stained with VEGFR2 antibody are shown. (G) Biotinylation-based analysis of cell-surface VEGFR2. Surface proteins labeled with the biotinylation reagent sulfo-NHS-SS–biotin were pulled down with streptavidin-Sepharose, and 5% of the total cell lysate and biotinylated cell-surface protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blot analysis with antibody against VEGFR2. (H) Quantification of band density for the cell-surface VEGFR2. Percentage is expressed as the change in surface VEGFR2 after BFA treatment (relative to initial levels). The percentage represents mean (± SD) for n = 3 and P ≤ .05. Scale bar represents 5 μm.

Article Snippet: The mAbs against early endosome-associated antigen 1 (EEA1), syntaxin 6, and trans-Golgi network 46 (TGN46), the rat anti–mouse CD31 antibody, and growth factor–reduced Matrigel were obtained from BD Biosciences.

Techniques: Labeling, Marker, Fluorescence, Generated, Gradient Centrifugation, Immunofluorescence, Imaging, Staining, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Blood

Article Title: Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6

doi: 10.1182/blood-2010-06-291690

Figure Lengend Snippet: Inhibition of syntaxin 6 function decreases the levels of VEGFR2 but not the levels of VEGFR1. (A,D) Uninfected (Control) and syntaxin 6-cyto or syntaxin 16-cyto expressing HUVECs (after 20 hours of infection) were stained with mAb against VEGFR2 (55B11), syntaxin 6 (in Control and syntaxin 6-cyto–treated cells) or syntaxin 16 (in syntaxin 16-cyto–treated cells). Samples were then fixed and observed under fluorescence microscope. (B) HUVECs were subjected to siRNA-mediated syntaxin 6 or syntaxin 16 knockdown, and immunostained for VEGFR2. Representative images showing staining for intracellular VEGFR2 in cells in which endogenous syntaxin 6 or syntaxin 16 was knocked-down over 90% after 72 hours of siRNA treatment. (C) Samples were fixed and observed as in panel A, but stained with goat pAb against VEGFR1 and antibodies against syntaxin 6 or syntaxin 16. (D) Quantification of intracellular VEGFR2 or VEGFR1 in syntaxin 6-cyto and syntaxin 16-cyto expressing cells, and in cells in which endogenous syntaxin was knocked down by siRNA treatment (as in panels A-C). Epifluorescence images were acquired and total cell-associated fluorescence was quantified by image analysis. Values represent relative change in the levels of VEGFR2 or VEGFR1 normalized to an arbitrary value of 100 for untreated controls. Percentage is expressed as mean (± SD) of n = 90 cells for each condition from 3 separate experiments; P ≤ .001. (E) Lysates were prepared from uninfected, syntaxin 6-cyto– or syntaxin16-cyto–infected HUVECs (after infection for various periods of time, as indicated) and samples were immunoblotted for VEGFR2 (55B11) and VEGFR1 (rabbit polyclonal). Relative level of endogenous syntaxin 6, syntaxin 16, or tubulin in cell lysate is shown. (F) VEGFR2 and VEGFR1 band density from panel E was quantified and results represent relative levels of VEGFR2 and VEGFR1 after normalization to an arbitrary value of 100 for 0 minutes after infection. Percentage is expressed as mean (± SD) for n = 3; P ≤ .005). Scale bar represents 5 μm.

Article Snippet: The mAbs against early endosome-associated antigen 1 (EEA1), syntaxin 6, and trans-Golgi network 46 (TGN46), the rat anti–mouse CD31 antibody, and growth factor–reduced Matrigel were obtained from BD Biosciences.

Techniques: Inhibition, Expressing, Infection, Staining, Fluorescence, Microscopy