pcaggs vector Search Results


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  • 90
    Addgene inc pcaggs vector
    Overexpression of <t>MARCKSL1</t> induces anxiety-like behaviors. (A) Schema of the genomic organization of Marcksl1 inserted into a <t>pCAGGS</t> vector. The arrows represent the positions of genotyping primers. The dotted line box indicates the primer sequences. The right red bands are the result of genotyping. (B) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (C) MARCKSL1 (green) staining in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test scores and elevated plus maze test scores in WT and Tg/Tg mice (each group, n = 11). Data are presented as means ± SEM. *p
    Pcaggs Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcaggs vector/product/Addgene inc
    Average 90 stars, based on 167 article reviews
    Price from $9.99 to $1999.99
    pcaggs vector - by Bioz Stars, 2020-07
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    88
    Thermo Fisher vector pcaggs
    Overexpression of <t>MARCKSL1</t> induces anxiety-like behaviors. (A) Schema of the genomic organization of Marcksl1 inserted into a <t>pCAGGS</t> vector. The arrows represent the positions of genotyping primers. The dotted line box indicates the primer sequences. The right red bands are the result of genotyping. (B) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (C) MARCKSL1 (green) staining in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test scores and elevated plus maze test scores in WT and Tg/Tg mice (each group, n = 11). Data are presented as means ± SEM. *p
    Vector Pcaggs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector pcaggs/product/Thermo Fisher
    Average 88 stars, based on 35 article reviews
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    vector pcaggs - by Bioz Stars, 2020-07
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    90
    Stratagene pcaggs vector
    Overexpression of <t>MARCKSL1</t> induces anxiety-like behaviors. (A) Schema of the genomic organization of Marcksl1 inserted into a <t>pCAGGS</t> vector. The arrows represent the positions of genotyping primers. The dotted line box indicates the primer sequences. The right red bands are the result of genotyping. (B) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (C) MARCKSL1 (green) staining in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test scores and elevated plus maze test scores in WT and Tg/Tg mice (each group, n = 11). Data are presented as means ± SEM. *p
    Pcaggs Vector, supplied by Stratagene, used in various techniques. Bioz Stars score: 90/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcaggs vector/product/Stratagene
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    pcaggs vector - by Bioz Stars, 2020-07
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    85
    Gene Bridges Inc pcaggs flp vector
    Overexpression of <t>MARCKSL1</t> induces anxiety-like behaviors. (A) Schema of the genomic organization of Marcksl1 inserted into a <t>pCAGGS</t> vector. The arrows represent the positions of genotyping primers. The dotted line box indicates the primer sequences. The right red bands are the result of genotyping. (B) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (C) MARCKSL1 (green) staining in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test scores and elevated plus maze test scores in WT and Tg/Tg mice (each group, n = 11). Data are presented as means ± SEM. *p
    Pcaggs Flp Vector, supplied by Gene Bridges Inc, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcaggs flp vector/product/Gene Bridges Inc
    Average 85 stars, based on 6 article reviews
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    pcaggs flp vector - by Bioz Stars, 2020-07
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    88
    TaKaRa pcaggs ha vector
    Mir-30c-5p inhibited the infection of PEDV by regulating the expression of <t>SOCS1.</t> (A) SOCS1 overexpression increased PEDV infection and undermined the anti-PEDV activity of IFN-λ. Vero E6 cells were transfected as described with <t>pCAGGS-HA,</t> pCAGGS-SOCS1, and miR-30c-5p for 24 h, followed by incubation with porcine IFN-λ (100 ng/ml) or DMEM for 12 h. The cells then were infected with PEDV at an MOI of 0.1; PEDV infection was determined at 36 hpi. (B,C) miR-30c-5p abolished the impairment of the overexpression of SOCS1 to IFN-λ signaling under IFN-λ-stimulated or PEDV-infected conditions. E6 cells were treated as described in the legend for panel A. The cells were collected for RT-qPCR analysis of IFIT1 and ISG15 expression relative to that of GAPDH. Error bars, mean ± SEM ( n = 3 independent experiments). * P
    Pcaggs Ha Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Gene Bridges Inc pcaggs flpe vector
    Mir-30c-5p inhibited the infection of PEDV by regulating the expression of <t>SOCS1.</t> (A) SOCS1 overexpression increased PEDV infection and undermined the anti-PEDV activity of IFN-λ. Vero E6 cells were transfected as described with <t>pCAGGS-HA,</t> pCAGGS-SOCS1, and miR-30c-5p for 24 h, followed by incubation with porcine IFN-λ (100 ng/ml) or DMEM for 12 h. The cells then were infected with PEDV at an MOI of 0.1; PEDV infection was determined at 36 hpi. (B,C) miR-30c-5p abolished the impairment of the overexpression of SOCS1 to IFN-λ signaling under IFN-λ-stimulated or PEDV-infected conditions. E6 cells were treated as described in the legend for panel A. The cells were collected for RT-qPCR analysis of IFIT1 and ISG15 expression relative to that of GAPDH. Error bars, mean ± SEM ( n = 3 independent experiments). * P
    Pcaggs Flpe Vector, supplied by Gene Bridges Inc, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 29 article reviews
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    pcaggs flpe vector - by Bioz Stars, 2020-07
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    90
    Addgene inc pcaggs expression vector
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Expression Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcaggs expression vector/product/Addgene inc
    Average 90 stars, based on 154 article reviews
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    pcaggs expression vector - by Bioz Stars, 2020-07
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    90
    Addgene inc pcaggs gfp vector
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Gfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcaggs gfp vector - by Bioz Stars, 2020-07
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    90
    Addgene inc pcaggs flpe vector
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Flpe Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcaggs flpe vector/product/Addgene inc
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    pcaggs flpe vector - by Bioz Stars, 2020-07
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    93
    TaKaRa pcaggs mcherry
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Mcherry, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcaggs mcherry - by Bioz Stars, 2020-07
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    92
    Genewiz pcaggs vector
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Vector, supplied by Genewiz, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcaggs vector - by Bioz Stars, 2020-07
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    90
    AstraZeneca pcaggs vector
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Vector, supplied by AstraZeneca, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcaggs vector - by Bioz Stars, 2020-07
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    93
    Addgene inc pcaggs vectors
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc vector pcaggs hs dicer1
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Vector Pcaggs Hs Dicer1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    St. Jude Children's Research Hospital pcaggs mcs vector
    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty <t>pCAGGS</t> vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with <t>anti-SARS-CoV</t> nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.
    Pcaggs Mcs Vector, supplied by St. Jude Children's Research Hospital, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcaggs mcs vector - by Bioz Stars, 2020-07
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    84
    Addgene inc pcaggs 3e5e egfp vector
    Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial <t>pCAGGS-HHRz-3M-eGFP-5M</t> minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.
    Pcaggs 3e5e Egfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Addgene inc vector pcaggs chr2 venus
    Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial <t>pCAGGS-HHRz-3M-eGFP-5M</t> minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.
    Vector Pcaggs Chr2 Venus, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    vector pcaggs chr2 venus - by Bioz Stars, 2020-07
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    91
    Addgene inc pcaggs egfp puro vector
    Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial <t>pCAGGS-HHRz-3M-eGFP-5M</t> minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.
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    Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial <t>pCAGGS-HHRz-3M-eGFP-5M</t> minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.
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    Addgene inc pcaggs flpe puro vector
    Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial <t>pCAGGS-HHRz-3M-eGFP-5M</t> minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.
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    Image Search Results


    Overexpression of MARCKSL1 induces anxiety-like behaviors. (A) Schema of the genomic organization of Marcksl1 inserted into a pCAGGS vector. The arrows represent the positions of genotyping primers. The dotted line box indicates the primer sequences. The right red bands are the result of genotyping. (B) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (C) MARCKSL1 (green) staining in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test scores and elevated plus maze test scores in WT and Tg/Tg mice (each group, n = 11). Data are presented as means ± SEM. *p

    Journal: EBioMedicine

    Article Title: MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors

    doi: 10.1016/j.ebiom.2018.03.018

    Figure Lengend Snippet: Overexpression of MARCKSL1 induces anxiety-like behaviors. (A) Schema of the genomic organization of Marcksl1 inserted into a pCAGGS vector. The arrows represent the positions of genotyping primers. The dotted line box indicates the primer sequences. The right red bands are the result of genotyping. (B) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (C) MARCKSL1 (green) staining in the amygdala (three areas surrounded by the dotted lines) in WT and Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test scores and elevated plus maze test scores in WT and Tg/Tg mice (each group, n = 11). Data are presented as means ± SEM. *p

    Article Snippet: The fragment was subcloned into the Xho I and Eco RI site of a pCAGGS vector (Addgene) by introducing Marcksl1 containing a HA tag ( A).

    Techniques: Over Expression, Plasmid Preparation, In Situ Hybridization, Mouse Assay, Staining

    Mir-30c-5p inhibited the infection of PEDV by regulating the expression of SOCS1. (A) SOCS1 overexpression increased PEDV infection and undermined the anti-PEDV activity of IFN-λ. Vero E6 cells were transfected as described with pCAGGS-HA, pCAGGS-SOCS1, and miR-30c-5p for 24 h, followed by incubation with porcine IFN-λ (100 ng/ml) or DMEM for 12 h. The cells then were infected with PEDV at an MOI of 0.1; PEDV infection was determined at 36 hpi. (B,C) miR-30c-5p abolished the impairment of the overexpression of SOCS1 to IFN-λ signaling under IFN-λ-stimulated or PEDV-infected conditions. E6 cells were treated as described in the legend for panel A. The cells were collected for RT-qPCR analysis of IFIT1 and ISG15 expression relative to that of GAPDH. Error bars, mean ± SEM ( n = 3 independent experiments). * P

    Journal: Frontiers in Microbiology

    Article Title: The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis

    doi: 10.3389/fmicb.2020.01180

    Figure Lengend Snippet: Mir-30c-5p inhibited the infection of PEDV by regulating the expression of SOCS1. (A) SOCS1 overexpression increased PEDV infection and undermined the anti-PEDV activity of IFN-λ. Vero E6 cells were transfected as described with pCAGGS-HA, pCAGGS-SOCS1, and miR-30c-5p for 24 h, followed by incubation with porcine IFN-λ (100 ng/ml) or DMEM for 12 h. The cells then were infected with PEDV at an MOI of 0.1; PEDV infection was determined at 36 hpi. (B,C) miR-30c-5p abolished the impairment of the overexpression of SOCS1 to IFN-λ signaling under IFN-λ-stimulated or PEDV-infected conditions. E6 cells were treated as described in the legend for panel A. The cells were collected for RT-qPCR analysis of IFIT1 and ISG15 expression relative to that of GAPDH. Error bars, mean ± SEM ( n = 3 independent experiments). * P

    Article Snippet: To construct the monkey SOCS1 expression vector, the full-length CDS region of monkey SOCS1 was amplified from Vero E6 cellular mRNA PCR and cloned into pCAGGS-HA vector (Clontech, Mountain View, CA, USA) using EcoR I and Kpn I restriction sites.

    Techniques: Infection, Expressing, Over Expression, Activity Assay, Transfection, Incubation, Quantitative RT-PCR

    SOCS1 counteracted the anti-PEDV activity of IFN-λ. (A) The knockdown efficiency of shSOCS1 was determined by Western blotting (left panel); β-actin was used as a loading control. The SOCS1 bands were quantified using ImageJ software as normalized to β-actin (right panel). (B) Knockdown of SOCS1 decreased PEDV replication. Vero E6 cells were treated with 100 ng/mL of IFN-λ and transfected with NC or shSOCS1s, followed by infection with PEDV (MOI = 0.1). After 36 h, cell culture supernatants were harvested for virus titration. (C) Silencing of SOCS1 increased IFN-λ signaling induced by IFN-λ stimulation. Vero E6 cells were stimulated with IFN-λ 12 h after transfection with shSOCS1 #1, shSOCS1 #2, shSOCS1 #3, or scrambled control shRNA, and then the cells were collected for RT-qPCR analysis of ISG15, IFIT1, or MxA expression relative to that of GAPDH after 24 h of stimulation. (D) Transient expression of SOCS1 in Vero E6 cells. SOCS1 was cloned and expressed in the eukaryotic expression vector pCAGGS-HA. The overexpression efficiency of SOCS1 in Vero E6 cells was confirmed by IFA. (E) Transient expression of SOCS1 enhanced PEDV infection in Vero E6 cells. Vero E6 cells were stimulated with IFN-λ 12 h after the transient expression of SOCS1 promoted PEDV infection. Vero E6 cells were transfected with SOCS1-HA for 24 h and then infected with PEDV (MOI = 0.1). PEDV infection was determined by measuring PEDV titers. (F) Transient expression of SOCS1 disrupted the IFN-λ antiviral response. Vero E6 cells were stimulated with IFN-λ after being transfected with SOCS1 for 24 h. The cells were collected for RT-qPCR analysis of ISG15, MxA, and IFIT1 expression relative to that of GAPDH after 24 h of stimulation. Error bars, mean ±SEM. ( n = 3 independent experiments). * P

    Journal: Frontiers in Microbiology

    Article Title: The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis

    doi: 10.3389/fmicb.2020.01180

    Figure Lengend Snippet: SOCS1 counteracted the anti-PEDV activity of IFN-λ. (A) The knockdown efficiency of shSOCS1 was determined by Western blotting (left panel); β-actin was used as a loading control. The SOCS1 bands were quantified using ImageJ software as normalized to β-actin (right panel). (B) Knockdown of SOCS1 decreased PEDV replication. Vero E6 cells were treated with 100 ng/mL of IFN-λ and transfected with NC or shSOCS1s, followed by infection with PEDV (MOI = 0.1). After 36 h, cell culture supernatants were harvested for virus titration. (C) Silencing of SOCS1 increased IFN-λ signaling induced by IFN-λ stimulation. Vero E6 cells were stimulated with IFN-λ 12 h after transfection with shSOCS1 #1, shSOCS1 #2, shSOCS1 #3, or scrambled control shRNA, and then the cells were collected for RT-qPCR analysis of ISG15, IFIT1, or MxA expression relative to that of GAPDH after 24 h of stimulation. (D) Transient expression of SOCS1 in Vero E6 cells. SOCS1 was cloned and expressed in the eukaryotic expression vector pCAGGS-HA. The overexpression efficiency of SOCS1 in Vero E6 cells was confirmed by IFA. (E) Transient expression of SOCS1 enhanced PEDV infection in Vero E6 cells. Vero E6 cells were stimulated with IFN-λ 12 h after the transient expression of SOCS1 promoted PEDV infection. Vero E6 cells were transfected with SOCS1-HA for 24 h and then infected with PEDV (MOI = 0.1). PEDV infection was determined by measuring PEDV titers. (F) Transient expression of SOCS1 disrupted the IFN-λ antiviral response. Vero E6 cells were stimulated with IFN-λ after being transfected with SOCS1 for 24 h. The cells were collected for RT-qPCR analysis of ISG15, MxA, and IFIT1 expression relative to that of GAPDH after 24 h of stimulation. Error bars, mean ±SEM. ( n = 3 independent experiments). * P

    Article Snippet: To construct the monkey SOCS1 expression vector, the full-length CDS region of monkey SOCS1 was amplified from Vero E6 cellular mRNA PCR and cloned into pCAGGS-HA vector (Clontech, Mountain View, CA, USA) using EcoR I and Kpn I restriction sites.

    Techniques: Activity Assay, Western Blot, Software, Transfection, Infection, Cell Culture, Titration, shRNA, Quantitative RT-PCR, Expressing, Clone Assay, Plasmid Preparation, Over Expression, Immunofluorescence

    PLSCR1 inhibits the nuclear import of NP. (A, B) Empty retrovirus-transduced control A549 cells (A) or PLSCR1-overexpressing A549 cells (B) were infected with WSN virus at an MOI of 5. At 4, 6, 8, 10, and 12 h p.i., the infected cells were fixed and stained with mouse anti-NP mAb and rabbit anti-PLSCR1 pAb, followed by incubation with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). The nuclei were stained with DAPI. (C) Quantitative analysis of NP localization in virus-infected cells. On the basis of the confocal microscopy in panels A and B, the localization of NP (indicative of vRNP) after nuclear import was categorized into four types, clear nuclear localization, simultaneous localization at the edge of the nucleus and the cytoplasm, predominant cytoplasmic localization, and close to the cytoplasmic membrane. The results shown are calculated from one hundred cells viewed under a confocal microscope with a 40X objective lens. (D). PLSCR1 inhibits the nuclear import of NP in transfected A549 cells. A549 cells were transfected with pCAGGS-WSNNP alone or were cotransfected with pCAGGS-WSNNP and pCAGGS-PLSCR1 and were assessed by confocal microscopy. NP was detected with a mouse anti-NP mAb and visualized with Alexa Fluor 633 (red). PLSCR1 was detected with a rabbit anti-PLSCR1 pAb and visualized with Alexa Fluor 488 (green). Yellow in the merged image indicates the colocalization of NP and PLSCR1. (E) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. At 6 h p.i., the cells were separated into nuclear (N) and cytoplasmic fractions (C). Each fraction was subjected to western blotting with a rabbit anti-NP pAb and a rabbit anti-PLSCR1 pAb for protein detection. (F) PLSCR1-overexpressing A549 cells or control A549 cells were treated with CHX to inhibit protein synthesis. The treated cells were infected with WSN virus at an MOI of 5, and were separated into nuclear and cytoplasmic fractions at 2 h p.i., followed by western blotting to detect the amount of NP in the nuclear and cytoplasmic fractions.

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: PLSCR1 inhibits the nuclear import of NP. (A, B) Empty retrovirus-transduced control A549 cells (A) or PLSCR1-overexpressing A549 cells (B) were infected with WSN virus at an MOI of 5. At 4, 6, 8, 10, and 12 h p.i., the infected cells were fixed and stained with mouse anti-NP mAb and rabbit anti-PLSCR1 pAb, followed by incubation with Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). The nuclei were stained with DAPI. (C) Quantitative analysis of NP localization in virus-infected cells. On the basis of the confocal microscopy in panels A and B, the localization of NP (indicative of vRNP) after nuclear import was categorized into four types, clear nuclear localization, simultaneous localization at the edge of the nucleus and the cytoplasm, predominant cytoplasmic localization, and close to the cytoplasmic membrane. The results shown are calculated from one hundred cells viewed under a confocal microscope with a 40X objective lens. (D). PLSCR1 inhibits the nuclear import of NP in transfected A549 cells. A549 cells were transfected with pCAGGS-WSNNP alone or were cotransfected with pCAGGS-WSNNP and pCAGGS-PLSCR1 and were assessed by confocal microscopy. NP was detected with a mouse anti-NP mAb and visualized with Alexa Fluor 633 (red). PLSCR1 was detected with a rabbit anti-PLSCR1 pAb and visualized with Alexa Fluor 488 (green). Yellow in the merged image indicates the colocalization of NP and PLSCR1. (E) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. At 6 h p.i., the cells were separated into nuclear (N) and cytoplasmic fractions (C). Each fraction was subjected to western blotting with a rabbit anti-NP pAb and a rabbit anti-PLSCR1 pAb for protein detection. (F) PLSCR1-overexpressing A549 cells or control A549 cells were treated with CHX to inhibit protein synthesis. The treated cells were infected with WSN virus at an MOI of 5, and were separated into nuclear and cytoplasmic fractions at 2 h p.i., followed by western blotting to detect the amount of NP in the nuclear and cytoplasmic fractions.

    Article Snippet: Truncation mutants of GST-tagged WSN NP were generated by using a PCR approach and were cloned into the pCAGGS vector. pQCXIN-PLSCR1 was constructed by inserting the PLSCR1 ORF into the pQCXIN vector (Clontech).

    Techniques: Infection, Staining, Incubation, Confocal Microscopy, Microscopy, Transfection, Western Blot

    PLSCR1 does not affect the phosphorylation status of NP and does not stimulate the IFN pathways. (A) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. Cell lysates were processed at 6 h and 8 h p.i., immunoprecipitated with a mouse anti-NP mAb, a mouse anti-p-Ser mAb, or a mouse anti-p-Tyr mAb, followed by western blotting with a rabbit anti-NP pAb to detect the level of total NP, serine-phosphorylated NP, and tyrosine-phosphorylated NP, respectively. (B) Replication of an NP-phosphorylation mutant virus in PLSCR1-overexpressing A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with wild-type WSN virus or the phosphorylation mutant S9A/Y10F at an MOI of 0.1. Supernatants were collected at the indicated timepoints and titrated for infectious virus by means of plaque assay on MDCK cells. (C) Expression of Mx1 protein in PLSCR1-overexpressing or control A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were grown in 12-well plates, and were left untreated or were treated with IFN-α for 24 h. The cell lysates were then subjected to western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. GAPDH, detected by a rabbit anti-GAPDH pAb, served as a negative control. (D) Expression of the ISRE luciferase reporter gene in HEK293T cells transfected with the PLSCR1-expressing construct or empty vector. HEK293T cells were transfected with the ISRE-Luc reporter plasmid, pRL-TK control plasmid, and the pCAGGS-PLSCR1 or empty pCAGGS plasmid for 20 h. The overexpression of PLSCR1 was confirmed by western blotting with a rabbit anti-PLSCR1 pAb. The luciferase activity of the transfected cells was analyzed by using the Dual-Luciferase reporter assay. After normalization with co-transfected Renilla luciferase activity, the relative firefly luciferase activity of PLSCR1-overexpressing cells was expressed as the fold-induction of the ISRE firefly luciferase activity compared to cells transfected with empty pCAGGS vector. **, P

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: PLSCR1 does not affect the phosphorylation status of NP and does not stimulate the IFN pathways. (A) PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with WSN virus at an MOI of 5. Cell lysates were processed at 6 h and 8 h p.i., immunoprecipitated with a mouse anti-NP mAb, a mouse anti-p-Ser mAb, or a mouse anti-p-Tyr mAb, followed by western blotting with a rabbit anti-NP pAb to detect the level of total NP, serine-phosphorylated NP, and tyrosine-phosphorylated NP, respectively. (B) Replication of an NP-phosphorylation mutant virus in PLSCR1-overexpressing A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were infected with wild-type WSN virus or the phosphorylation mutant S9A/Y10F at an MOI of 0.1. Supernatants were collected at the indicated timepoints and titrated for infectious virus by means of plaque assay on MDCK cells. (C) Expression of Mx1 protein in PLSCR1-overexpressing or control A549 cells. PLSCR1-overexpressing or empty retrovirus-transduced control A549 cells were grown in 12-well plates, and were left untreated or were treated with IFN-α for 24 h. The cell lysates were then subjected to western blotting with a rabbit anti-Mx1 pAb for the detection of Mx1 protein. GAPDH, detected by a rabbit anti-GAPDH pAb, served as a negative control. (D) Expression of the ISRE luciferase reporter gene in HEK293T cells transfected with the PLSCR1-expressing construct or empty vector. HEK293T cells were transfected with the ISRE-Luc reporter plasmid, pRL-TK control plasmid, and the pCAGGS-PLSCR1 or empty pCAGGS plasmid for 20 h. The overexpression of PLSCR1 was confirmed by western blotting with a rabbit anti-PLSCR1 pAb. The luciferase activity of the transfected cells was analyzed by using the Dual-Luciferase reporter assay. After normalization with co-transfected Renilla luciferase activity, the relative firefly luciferase activity of PLSCR1-overexpressing cells was expressed as the fold-induction of the ISRE firefly luciferase activity compared to cells transfected with empty pCAGGS vector. **, P

    Article Snippet: Truncation mutants of GST-tagged WSN NP were generated by using a PCR approach and were cloned into the pCAGGS vector. pQCXIN-PLSCR1 was constructed by inserting the PLSCR1 ORF into the pQCXIN vector (Clontech).

    Techniques: Infection, Immunoprecipitation, Western Blot, Mutagenesis, Plaque Assay, Expressing, Negative Control, Luciferase, Transfection, Construct, Plasmid Preparation, Over Expression, Activity Assay, Reporter Assay

    NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend Snippet: NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

    Article Snippet: Truncation mutants of GST-tagged WSN NP were generated by using a PCR approach and were cloned into the pCAGGS vector. pQCXIN-PLSCR1 was constructed by inserting the PLSCR1 ORF into the pQCXIN vector (Clontech).

    Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads

    MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty pCAGGS vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with anti-SARS-CoV nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.

    Journal: mBio

    Article Title: Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication

    doi: 10.1128/mBio.01658-17

    Figure Lengend Snippet: MERS-CoV nsp3 and nsp4 interact with each other. (A) Scaled schematic overview of MERS-CoV pp1ab and nsp3-4 constructs. Amino acid numbers refer to the MERS-CoV pp1ab sequence. The expected cleavage of the nsp3/nsp4 junction by PL pro is indicated. The epitope tags used at the termini of the constructs are indicated with ovals. TM, transmembrane region. (B) 293T cells were transfected with MERS-CoV nsp3-4 plasmids or empty pCAGGS vector (EV) and analyzed by Western blotting 20 h posttransfection. nsp3 was detected with anti-SARS-CoV nsp3 serum that cross-reacts with MERS-CoV nsp3 ( 21 ), and nsp4 was detected with anti-V5 monoclonal antibody. (C) Constructs expressing MERS-CoV nsp3 or nsp4 or a GFP control were transfected into 293T cells, which were metabolically labeled with [ 35 S]methionine-cysteine from 4 to 20 h posttransfection. Lysates were immunoprecipitated with the indicated antibodies, separated on an SDS-PAGE gel, and visualized using phosphorimaging. Bands not corresponding to expected protein size in the Western blot are indicated with asterisks. The ~130-kDa band in the nsp3 IP was also observed in the Western blot. nsp4 bands in IP were fuzzy likely due to the relatively high hydrophobicity of the protein. (D) HuH-7 cells were transfected with the indicated plasmids, and localization of MERS-CoV nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-SARS-CoV-nsp3 serum, and nsp4 was detected with anti-V5 monoclonal antibody.

    Article Snippet: Coding sequences were transferred to the pCAGGS expression vector (Addgene) for expression. pCAGGS-SARS-nsp4 was described previously ( ).

    Techniques: Construct, Sequencing, Transfection, Plasmid Preparation, Western Blot, Expressing, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Immunofluorescence, Confocal Microscopy

    Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial pCAGGS-HHRz-3M-eGFP-5M minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.

    Journal: bioRxiv

    Article Title: Development and optimization of a novel T7 polymerase-independent Marburg virus minigenome system

    doi: 10.1101/2020.05.11.088211

    Figure Lengend Snippet: Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in the initial pCAGGS-HHRz-3M-eGFP-5M minigenome. The arrow indicates the position at which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is indicated with an arrow, while the residues marked in black correspond to the nucleotides incorporated into stem I of the hammerhead ribozyme.

    Article Snippet: Each of the resulting MARV minigenomes was then cloned into the pCAGGS_3E5E_eGFP vector, which was a gift from Elke Mühlberger (Addgene plasmid # 103054).

    Techniques: Sequencing

    Shortening the ribozyme stem increases minigenome activity. (A) Transfection of HEK293T cells with different mutants of the pCAGGS-HHRz-3M-eGFP-5M minigenome shows that minigenome activity can be increased by reducing complementarity between the ribozyme and the MARV trailer. Shortening by 3-5 nucleotides significantly increases activity, with an optimum at a residual stem length of six base pairs. Data from three independent experiments, done in triplicate. Error bars indicate the standard error of mean. Significance testing was done using one-way ANOVA with Dunnett’s test for multiple comparisons: * p

    Journal: bioRxiv

    Article Title: Development and optimization of a novel T7 polymerase-independent Marburg virus minigenome system

    doi: 10.1101/2020.05.11.088211

    Figure Lengend Snippet: Shortening the ribozyme stem increases minigenome activity. (A) Transfection of HEK293T cells with different mutants of the pCAGGS-HHRz-3M-eGFP-5M minigenome shows that minigenome activity can be increased by reducing complementarity between the ribozyme and the MARV trailer. Shortening by 3-5 nucleotides significantly increases activity, with an optimum at a residual stem length of six base pairs. Data from three independent experiments, done in triplicate. Error bars indicate the standard error of mean. Significance testing was done using one-way ANOVA with Dunnett’s test for multiple comparisons: * p

    Article Snippet: Each of the resulting MARV minigenomes was then cloned into the pCAGGS_3E5E_eGFP vector, which was a gift from Elke Mühlberger (Addgene plasmid # 103054).

    Techniques: Activity Assay, Transfection

    Exchange of the T7 promoter by a mammalian CAG promotor leads to a significant loss of MARV minigenome activity. (A) Schematic representation of the pCAGGS-HHRz-3M-eGFP-5M minigenome, made by replacing the T7 promoter of the T7-3M-eGFP-5M plasmid by a CAG promoter and a 5’ hammerhead ribozyme (HHRz). HdVRz = hepatitis delta virus ribozyme. (B) Comparison of the T7 polymerase and RNA polymerase II eGFP minigenome systems. HEK293T cells were transfected with 1000 ng of the indicated minigenome, as well as all necessary support plasmids. Images were taken 48 hours post transfection. Cells are stained with Hoechst 33342 as background staining (shown in blue), eGFP-expressing cells are shown in green.

    Journal: bioRxiv

    Article Title: Development and optimization of a novel T7 polymerase-independent Marburg virus minigenome system

    doi: 10.1101/2020.05.11.088211

    Figure Lengend Snippet: Exchange of the T7 promoter by a mammalian CAG promotor leads to a significant loss of MARV minigenome activity. (A) Schematic representation of the pCAGGS-HHRz-3M-eGFP-5M minigenome, made by replacing the T7 promoter of the T7-3M-eGFP-5M plasmid by a CAG promoter and a 5’ hammerhead ribozyme (HHRz). HdVRz = hepatitis delta virus ribozyme. (B) Comparison of the T7 polymerase and RNA polymerase II eGFP minigenome systems. HEK293T cells were transfected with 1000 ng of the indicated minigenome, as well as all necessary support plasmids. Images were taken 48 hours post transfection. Cells are stained with Hoechst 33342 as background staining (shown in blue), eGFP-expressing cells are shown in green.

    Article Snippet: Each of the resulting MARV minigenomes was then cloned into the pCAGGS_3E5E_eGFP vector, which was a gift from Elke Mühlberger (Addgene plasmid # 103054).

    Techniques: Activity Assay, Plasmid Preparation, Transfection, Staining, Expressing