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: Aging Cell

Article Title: Extra virgin olive oil improves synaptic activity, short‐term plasticity, memory, and neuropathology in a tauopathy model

doi: 10.1111/acel.13076

Figure Lengend Snippet: Effect of chronic administration of EVOO‐rich diet on hippocampal pre‐ and postsynaptic protein expression in hTau mice. (a) Representative Western blot analyses for SNAP25, syntaxin 1A, synaptobrevin, complexin 1/2, and synaptophysin (SYP) in hippocampus homogenates from CTR and EVOO mice. (b) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (** p < .01; n = 6 per group) . (c) Representative Western blot analyses for the AMPAR subunit glutamate receptor 1 (GluR1), AMPAR subunit glutamate receptor 2 (GluR2), and their phosphorylated forms, pGluR1 (S831), pGluR1 (S845), and pGluR2 (S880), in hippocampus homogenates from CTR and EVOO mice. (d) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A ( n = 6 per group) . Values are mean ± SEM

Article Snippet: Syntaxin 1A , AF7237 , RD SYSTEMS , Goat.

Techniques: Expressing, Western Blot

Journal: Aging Cell

Article Title: Extra virgin olive oil improves synaptic activity, short‐term plasticity, memory, and neuropathology in a tauopathy model

doi: 10.1111/acel.13076

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: Syntaxin 1A , AF7237 , RD SYSTEMS , Goat.

Techniques:

Journal: mAbs

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

doi: 10.1080/19420862.2018.1551675

Figure Lengend 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.

Article Snippet: 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.

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

Journal: mAbs

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

doi: 10.1080/19420862.2018.1551675

Figure Lengend 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.

Article Snippet: 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.

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

Journal: mAbs

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

doi: 10.1080/19420862.2018.1551675

Figure Lengend 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.

Article Snippet: 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.

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

Journal: mAbs

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

doi: 10.1080/19420862.2018.1551675

Figure Lengend 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).

Article Snippet: 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.

Techniques: Microscopy, Staining

Journal: mAbs

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

doi: 10.1080/19420862.2018.1551675

Figure Lengend 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.

Article Snippet: 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.

Techniques: Cell Culture, Staining, Control, Incubation