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syntaxin 4  (Santa Cruz Biotechnology)


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    Structured Review

    Santa Cruz Biotechnology syntaxin 4
    Syntaxin 4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/syntaxin/Syntaxin+4+Antibody/pm11208155-200-19-40
    Average 92 stars, based on 27 article reviews
    syntaxin 4 - by Bioz Stars, 2026-09
    92/100 stars

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    Related Articles

    Blocking Assay:

    Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells
    Article Snippet: Slides were treated with 50–70 μl blocking solution (5% normal donkey serum (NDS, ImmunoReagents, SP-072-VX10), 1% BSA (Sigma-Aldrich, A1595-50ML), 8.5 ml PBS) in a humidified chamber at room temperature for 30 min. .. Blocking solution was replaced with antibody solution consisting of blocking solution with antibodies as follows: mCherry (Invitrogen, M11217, clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (for in vivo staining R&D Systems, MAB1262, clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082, clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301, clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, lot: L1916) 1:500, NeuN (Millipore, MAB377 clone A60) 1:500, S100a6 (Abcam, ab181975, clone EPR13084-69) 1:500, Tuj1 (BioLegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862, clone EPR4007-104) 1:100, calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signaling Technology, 4604) 1:500. .. We tested two mCherry antibodies (Abcam, ab213511 clone EPR20579; Invitrogen, M11217 clone 16D7) and we found that Invitrogen, M11217 was better for immunostaining for brain sections.

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: Slides were treated with 50–70 μl blocking solution (5% normal donkey serum (NDS, ImmunoReagents, SP-072-VX10), 1% BSA (Sigma-Aldrich, A1595-50ML), 8.5 ml PBS) in a humidified chamber at room temperature for 30 min. .. Blocking solution was replaced with antibody solution consisting of blocking solution with antibodies as follows: mCherry (Invitrogen, M11217, clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (for in vivo staining R&D Systems, MAB1262, clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082, clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301, clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, lot: L1916) 1:500, NeuN (Millipore, MAB377 clone A60) 1:500, S100a6 (Abcam, ab181975, clone EPR1308469) 1:500, Tuj1 (BioLegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862, clone EPR4007-104) 1:100, calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signaling Technology, 4604) 1:500. .. We tested two mCherry antibodies (Abcam, ab213511 clone EPR20579; Invitrogen, M11217 clone 16D7) and we found that Invitrogen, M11217 was better for immunostaining for brain sections.

    In Vivo:

    Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells
    Article Snippet: Slides were treated with 50–70 μl blocking solution (5% normal donkey serum (NDS, ImmunoReagents, SP-072-VX10), 1% BSA (Sigma-Aldrich, A1595-50ML), 8.5 ml PBS) in a humidified chamber at room temperature for 30 min. .. Blocking solution was replaced with antibody solution consisting of blocking solution with antibodies as follows: mCherry (Invitrogen, M11217, clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (for in vivo staining R&D Systems, MAB1262, clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082, clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301, clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, lot: L1916) 1:500, NeuN (Millipore, MAB377 clone A60) 1:500, S100a6 (Abcam, ab181975, clone EPR13084-69) 1:500, Tuj1 (BioLegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862, clone EPR4007-104) 1:100, calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signaling Technology, 4604) 1:500. .. We tested two mCherry antibodies (Abcam, ab213511 clone EPR20579; Invitrogen, M11217 clone 16D7) and we found that Invitrogen, M11217 was better for immunostaining for brain sections.

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: Slides were treated with 50–70 μl blocking solution (5% normal donkey serum (NDS, ImmunoReagents, SP-072-VX10), 1% BSA (Sigma-Aldrich, A1595-50ML), 8.5 ml PBS) in a humidified chamber at room temperature for 30 min. .. Blocking solution was replaced with antibody solution consisting of blocking solution with antibodies as follows: mCherry (Invitrogen, M11217, clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (for in vivo staining R&D Systems, MAB1262, clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082, clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301, clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, lot: L1916) 1:500, NeuN (Millipore, MAB377 clone A60) 1:500, S100a6 (Abcam, ab181975, clone EPR1308469) 1:500, Tuj1 (BioLegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862, clone EPR4007-104) 1:100, calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signaling Technology, 4604) 1:500. .. We tested two mCherry antibodies (Abcam, ab213511 clone EPR20579; Invitrogen, M11217 clone 16D7) and we found that Invitrogen, M11217 was better for immunostaining for brain sections.

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500. .. Immunofluorescence straining in brain sections in vivo: mCherry (Invitrogen, M11217 clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (R&D Systems, MAB1262 clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301 clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, Lot: L1916) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21202, A21206, A21209, A21447, A31571, A31573) 1:500, NeuN (Millipore, MAB377, clone A60) 1:500, S100a6 (Abcam, ab181975 clone EPR13084-69) 1:500, Tuj1 (Biolegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862 clone EPR4007-104) 1:100, Calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signalling Technology, 4604) 1:500.

    Staining:

    Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells
    Article Snippet: Slides were treated with 50–70 μl blocking solution (5% normal donkey serum (NDS, ImmunoReagents, SP-072-VX10), 1% BSA (Sigma-Aldrich, A1595-50ML), 8.5 ml PBS) in a humidified chamber at room temperature for 30 min. .. Blocking solution was replaced with antibody solution consisting of blocking solution with antibodies as follows: mCherry (Invitrogen, M11217, clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (for in vivo staining R&D Systems, MAB1262, clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082, clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301, clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, lot: L1916) 1:500, NeuN (Millipore, MAB377 clone A60) 1:500, S100a6 (Abcam, ab181975, clone EPR13084-69) 1:500, Tuj1 (BioLegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862, clone EPR4007-104) 1:100, calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signaling Technology, 4604) 1:500. .. We tested two mCherry antibodies (Abcam, ab213511 clone EPR20579; Invitrogen, M11217 clone 16D7) and we found that Invitrogen, M11217 was better for immunostaining for brain sections.

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: Slides were treated with 50–70 μl blocking solution (5% normal donkey serum (NDS, ImmunoReagents, SP-072-VX10), 1% BSA (Sigma-Aldrich, A1595-50ML), 8.5 ml PBS) in a humidified chamber at room temperature for 30 min. .. Blocking solution was replaced with antibody solution consisting of blocking solution with antibodies as follows: mCherry (Invitrogen, M11217, clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (for in vivo staining R&D Systems, MAB1262, clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082, clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301, clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, lot: L1916) 1:500, NeuN (Millipore, MAB377 clone A60) 1:500, S100a6 (Abcam, ab181975, clone EPR1308469) 1:500, Tuj1 (BioLegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862, clone EPR4007-104) 1:100, calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signaling Technology, 4604) 1:500. .. We tested two mCherry antibodies (Abcam, ab213511 clone EPR20579; Invitrogen, M11217 clone 16D7) and we found that Invitrogen, M11217 was better for immunostaining for brain sections.

    other:

    Article Title: α-Synuclein Deletion Impairs Platelet Function: A Role for SNARE Complex Assembly
    Article Snippet: FITC-labelled CD41 antibody (553848) was purchased from BD Pharmingen (Franklin Lakes, NJ, USA). α-synuclein (sc-515879), P-α-synuclein (ser129, sc-135638), syntaxin 11 (sc-377121), syntaxin 4 (sc-101301), and SNAP-23 (sc-373743) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Incubation:

    Article Title: Alpha-Taxilin: A Potential Diagnosis and Therapeutics Target in Rheumatoid Arthritis Which Interacts with Key Glycolytic Enzymes Associated with Metabolic Shifts in Fibroblast-Like Synoviocytes
    Article Snippet: The lysate was centrifuged (15,000 × g, 4°C, 30 min), and protein 40 microgram (μg) estimated by bicinchoninic acid assay (BCA) (G-biosciences, USA) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (NC) (G-biosciences, USA) using a semi dry transfer unit (Bio-Rad, USA). .. The membranes were then incubated (4°C overnight) with 5% Bovine Serum Albumin (BSA), followed by washing and incubation (overnight at 4°C) separately with diluted (1:3000) primary antibodies; Anti-p-65 (Santa Cruz, USA), anti-α-Taxilin (Santa Cruz, USA), anti- glyceraldehyde3-phosphate dehydrogenase (GAPDH) (Santa Cruz, USA), anti-STX-3 (Santa Cruz, USA), anti-STX-4 (Santa Cruz, USA), anti-TLR-4 (Cloud-Clone, USA), and anti-TLR-2 (Cloud-Clone, USA). .. Each NC membrane was then incubated for 3h at room temperature (RT) except for TLR-2 and TLR-4 antibodies, which were incubated overnight at 4°C as standardized.31 The membrane was washed and incubated (1h, RT) with HRP-conjugated anti-mouse secondary antibody (1:6000) (Jackson ImmunoResearch, USA), developed with an enhanced chemiluminescence reagent (Cyanogen, USA) obtained from Chemidoc MP (Bio-Rad, USA), and analyzed using Image Lab software (Bio-Rad, USA).16 Ribose Nucleic Acid Isolation and Quantitative Reverse Transcription Polymerase Chain Reaction For quantitative reverse transcription polymerase chain reaction (qRT-PCR), the cells were lysed with 500μl TRIzol (G-bioscience, USA), incubated (5min, RT), added 200μl chloroform (MercUSA), and then centrifuged (15min, 11,000 × g, 4°C).

    Article Title: Alpha-Taxilin: A Potential Diagnosis and Therapeutics Target in Rheumatoid Arthritis Which Interacts with Key Glycolytic Enzymes Associated with Metabolic Shifts in Fibroblast-Like Synoviocytes
    Article Snippet: The lysate was centrifuged (15,000 × g, 4°C, 30 min), and protein 40 microgram (μg) estimated by bicinchoninic acid assay (BCA) (G-biosciences, USA) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (NC) (G-biosciences, USA) using a semi dry transfer unit (Bio-Rad, USA). .. The membranes were then incubated (4°C overnight) with 5% Bovine Serum Albumin (BSA), followed by washing and incubation (overnight at 4°C) separately with diluted (1:3000) primary antibodies; Anti-p-65 (Santa Cruz, USA), anti-α-Taxilin (Santa Cruz, USA), anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz, USA), anti-STX-3 (Santa Cruz, USA), anti-STX-4 (Santa Cruz, USA), anti-TLR-4 (Cloud-Clone, USA), and anti-TLR-2 (Cloud-Clone, USA). ..

    Article Title: Carbon Dot-Loaded Apoptotic Vesicles Improve the Liver Kupffer Cell-Mediated Antibacterial Effect to Synergistically Alleviate Sepsis.
    Article Snippet: Sepsis is a lethal systemic inflammatory disease against infection that lacks effective therapeutic approaches.. Liver resident macrophage Kupffer cell (KC)-initiated bacterial clearance is crucial for the host to defend against infection.. However, it remains unclear whether this process also governs the antibacterial therapy of sepsis that would be used to improve therapeutic outcomes.

    Chromatin Immunoprecipitation:

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: .. 3 nature portfolio | reporting sum m ary April 2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence: In vitro: GLUT4 (Abcam, 33780) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, SC101301 clone QQ-17) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500.

    Flow Cytometry:

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: .. 3 nature portfolio | reporting sum m ary April 2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence: In vitro: GLUT4 (Abcam, 33780) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, SC101301 clone QQ-17) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500.

    Magnetic Resonance Imaging:

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: .. 3 nature portfolio | reporting sum m ary April 2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence: In vitro: GLUT4 (Abcam, 33780) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, SC101301 clone QQ-17) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500.

    Immunofluorescence:

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: .. 3 nature portfolio | reporting sum m ary April 2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence: In vitro: GLUT4 (Abcam, 33780) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, SC101301 clone QQ-17) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500.

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500. .. Immunofluorescence straining in brain sections in vivo: mCherry (Invitrogen, M11217 clone 16D7) 1:500, GFAP (Abcam, 53554) 1:500, GFP (Abcam, 13970) 1:500, GLUT4 (R&D Systems, MAB1262 clone 1F8) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, sc-101301 clone QQ-17) 1:500, GFP (Abcam, 13970) 1:500, mouse IgG (Santa Cruz SC-3877, Lot: L1916) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21202, A21206, A21209, A21447, A31571, A31573) 1:500, NeuN (Millipore, MAB377, clone A60) 1:500, S100a6 (Abcam, ab181975 clone EPR13084-69) 1:500, Tuj1 (Biolegend, 802001) 1:500, Olig2 (R&D Systems, AF2418) 1:100, Sox10 (Abcam, Ab180862 clone EPR4007-104) 1:100, Calretinin (Abcam, Ab244299) 1:500, Dcx (Cell Signalling Technology, 4604) 1:500.

    In Vitro:

    Article Title: CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.
    Article Snippet: .. 3 nature portfolio | reporting sum m ary April 2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence: In vitro: GLUT4 (Abcam, 33780) 1:500, Ki67 (Invitrogen, 14-5698-082 clone SolA15) 1:500, STX4A (Santa Cruz Biotechnology, SC101301 clone QQ-17) 1:500. .. Alexa 488/594/647 conjugated antibodies, (Fisher Scientific, A21206, A21209, A31571) 1:500.



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    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.

    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