recombinant h1n1 ha  (Sino Biological)


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  • 99
    Name:
    Influenza A H1N1 Hemagglutinin HA Protein
    Description:
    A DNA sequence encoding the extracellular domain of Influenza A virus A Brisbane 59 2007 H1N1 ACA28844 1 hemagglutinin Met 1 Gln 528 Native HA1 HA2 uncleaved was expressed with a C terminal polyhistidine tag
    Catalog Number:
    11052-V08H
    Price:
    None
    Category:
    recombinant protein
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological recombinant h1n1 ha
    A DNA sequence encoding the extracellular domain of Influenza A virus A Brisbane 59 2007 H1N1 ACA28844 1 hemagglutinin Met 1 Gln 528 Native HA1 HA2 uncleaved was expressed with a C terminal polyhistidine tag
    https://www.bioz.com/result/recombinant h1n1 ha/product/Sino Biological
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant h1n1 ha - by Bioz Stars, 2021-06
    99/100 stars

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    Related Articles

    Recombinant:

    Article Title: Cell-cultured, live attenuated, X-31ca-based H5N1 pre-pandemic influenza vaccine.
    Article Snippet: The HA and NA of the reassortant virus were derived from A/ Indonesia/5/2005 (H5N1), and the six internal genes from X-31ca (Lee et al., 2006). .. The recombinant HA protein of A/Indonesia/05/ 2005 produced from human HEK293 cell was purchased from Sino Biological (China). .. Optimization of vaccine production in cell cultureMDCK and Vero cells were infected with various titers of rH5N1ca and the cells were maintained in MEM supplemented by 5 µg/ml of trypsin and incubated at 30 °C in 5% CO2.

    Article Title: Rapid, Sensitive, and Selective Detection of H5 Hemagglutinin from Avian Influenza Virus Using an Immunowall Device
    Article Snippet: Phosphate-buffered saline (PBS; pH 7.4) solution and 9H -(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate) diammonium salt were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA). .. Mouse anti-H5N1 HA monoclonal antibody, recombinant H5N1 HA, recombinant H1N1 HA, recombinant H3N2 HA, recombinant H7N9 HA, and rabbit anti-avian influenza A HA polyclonal antibody were purchased from Sino Biological, Inc. (Beijing, China). .. DyLight 650-labeled goat antirabbit polyclonal antibody and alkaline phosphatase-labeled goat antirabbit polyclonal antibody were purchased from Abcam (Cambridge, MA).

    Article Title: Single-Step Detection of the Influenza Virus Hemagglutinin Using Bacterially-Produced Quenchbodies
    Article Snippet: Anti DYKDDDDK-tag antibody beads and the DYKDDDDK peptide were obtained from Wako Pure Chemicals (Osaka, Japan). .. The recombinant HA protein from A/California/04/2009 H1N1 was obtained from Sino Biological (Beijing, China). ..

    Article Title: Inhibition of influenza A virus infection by ginsenosides
    Article Snippet: For the determination of viral loads, lung tissues were collected on days 3 and 6 post infection (p.i.) from those animals who did not reach to the humane endpoint of 20% loss in body weight [ ]. .. Immunoblot assayDifferent concentrations of ginsenosides Rb1 were mixed with recombinant HA protein -Influenza A virus H1N1 (A/California/07/2009) (Sino Biological Inc., Beijing, China) and incubated at 37°C for 1 hour and then was spotted onto PVDF membrane. .. Immunoblotting was performed with Influenza A virus HA antibody (Santa cruz) and secondary goat anti-mouse IgG peroxidase conjugate (Calbiochem) and visualized by enhanced chemiluminescence (ECL).

    Article Title: A novel DNA vaccine expressing the Ag85A-HA2 fusion protein provides protection against influenza A virus and Staphylococcus aureus
    Article Snippet: Cells were segregated, washed, adjusted to a concentration of 5 × 106 cells per mL, and grown in a 96-well flat-bottom plate (5 × 105 cells per well) in RPMI 1640 medium, supplemented with HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), glutamine and 10% heat-inactivated fetal calf serum (FCS). .. Cells were stimulated with 0.5 μg concanavalin A (ConA) or 1.25 μg recombinant HA of A/PR/8/34 (Catalog Number: 11684-V08H, Sino Biological Inc, china) separately. .. Cells were incubated at 37°C in a humidified CO2 incubator, and supernatants were harvested after 72 h. Supernatants were pooled and stored frozen at −20°C until assay.

    Article Title: On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes.
    Article Snippet: .. The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. ..

    Produced:

    Article Title: Cell-cultured, live attenuated, X-31ca-based H5N1 pre-pandemic influenza vaccine.
    Article Snippet: The HA and NA of the reassortant virus were derived from A/ Indonesia/5/2005 (H5N1), and the six internal genes from X-31ca (Lee et al., 2006). .. The recombinant HA protein of A/Indonesia/05/ 2005 produced from human HEK293 cell was purchased from Sino Biological (China). .. Optimization of vaccine production in cell cultureMDCK and Vero cells were infected with various titers of rH5N1ca and the cells were maintained in MEM supplemented by 5 µg/ml of trypsin and incubated at 30 °C in 5% CO2.

    Purification:

    Article Title: Humanized antibody neutralizing 2009 pandemic H1N1 virus.
    Article Snippet: The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. .. The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. .. The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide.

    Incubation:

    Article Title: Inhibition of influenza A virus infection by ginsenosides
    Article Snippet: For the determination of viral loads, lung tissues were collected on days 3 and 6 post infection (p.i.) from those animals who did not reach to the humane endpoint of 20% loss in body weight [ ]. .. Immunoblot assayDifferent concentrations of ginsenosides Rb1 were mixed with recombinant HA protein -Influenza A virus H1N1 (A/California/07/2009) (Sino Biological Inc., Beijing, China) and incubated at 37°C for 1 hour and then was spotted onto PVDF membrane. .. Immunoblotting was performed with Influenza A virus HA antibody (Santa cruz) and secondary goat anti-mouse IgG peroxidase conjugate (Calbiochem) and visualized by enhanced chemiluminescence (ECL).

    Expressing:

    Article Title: On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes.
    Article Snippet: .. The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. ..

    Sequencing:

    Article Title: On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes.
    Article Snippet: .. The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. ..

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  • 92
    Sino Biological rabbit anti ha polyclonal antibody
    M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag <t>polyclonal</t> antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.
    Rabbit Anti Ha Polyclonal Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti ha polyclonal antibody/product/Sino Biological
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ha polyclonal antibody - by Bioz Stars, 2021-06
    92/100 stars
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    94
    Sino Biological influenza a virus hemagglutinin ha antibody rabbit mab
    Effect of λ-CGN on <t>influenza</t> A infection in vivo . BALB/c mice were mock-infected (black) or intranasally infected with maPR8 at 5 MLD50 (red). As test groups, the virus was preincubated at room temperature for 30 min with λ-CGN at a lower dose (1 mg/kg/d, bright green) or a higher dose (5 mg/kg/d, dark green), followed by intranasal administration. Control mice received oral OSV-P twice a day (10 mg/kg/d) at 8-h intervals, starting at 4 h before viral infection (blue). Body weight (A) and mortality (B) of mice were measured every day from Days 0 to 14 post-infection. Data are expressed as the mean ± SEM from five mice
    Influenza A Virus Hemagglutinin Ha Antibody Rabbit Mab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/influenza a virus hemagglutinin ha antibody rabbit mab/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    influenza a virus hemagglutinin ha antibody rabbit mab - by Bioz Stars, 2021-06
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    94
    Sino Biological pr8
    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 <t>PR8.</t> Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p
    Pr8, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pr8/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 - by Bioz Stars, 2021-06
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    80
    Sino Biological recombinant mex4108 ha protein plasma
    Validated miRNA targets are involved in immune response and cell proliferation in the lungs. Expression levels of (A) KRAS , BLIMP1 , and CDK6 (targets of let-7f); (B) SIRT1 , ZAP70 , and MYC (targets of miR-34c); (C) CAMTA1 (target of miR-129); and (D) PTEN (target of miR-18b) in BAL from rhesus macaques (five aged and three young adults) following <t>MEX4108</t> infection are shown. Expression of mRNA was normalized to expression of RPL32 . Changes in gene expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. (A–D) *let-7f, miR-34c, miR-129, miR-18-b, KRAS , SIRT1 , CAMTA1 , and PTEN ; ‡ KRAS and ZAP70 ; † CDK6 and MYC . * p
    Recombinant Mex4108 Ha Protein Plasma, supplied by Sino Biological, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant mex4108 ha protein plasma/product/Sino Biological
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Incubation, Purification, Staining, Immunofluorescence, Co-Immunoprecipitation Assay

    A leucine residue at position 96 of M2 is required for the TRAPPC6A interaction. (A) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1, pEGFP-C1-SC09 M2, pEGFP-C1-SC09 M2EDTM, or pEGFP-C1-SC09 M2CT into HEK293T cells for 48 h before preparation for cell lysates. Following immunoprecipitation with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody and a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (B and C) Plasmids expressing TRAPPC6A-myc and Flag-SC09M2 or Flag-SC09M2 with different amino acid deletions in the C terminus were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: A leucine residue at position 96 of M2 is required for the TRAPPC6A interaction. (A) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1, pEGFP-C1-SC09 M2, pEGFP-C1-SC09 M2EDTM, or pEGFP-C1-SC09 M2CT into HEK293T cells for 48 h before preparation for cell lysates. Following immunoprecipitation with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody and a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (B and C) Plasmids expressing TRAPPC6A-myc and Flag-SC09M2 or Flag-SC09M2 with different amino acid deletions in the C terminus were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Immunoprecipitation, Western Blot, Expressing

    Dynamics of the interaction of M2 and TRAPPC6AΔ in wt and mutant WSN virus-infected cells. A549 cells were infected with wt influenza virus WSN (A), or one of the M2 deletion mutants WSN M2Del1 (B) and WSN M2Del2 (C), at an MOI of 5. At 4, 6, 8, 10, and 14 h p.i., the infected cells were fixed and stained with mouse anti-M2 MAb 14C2 and rabbit anti-TRAPPC6A polyclonal antibody, followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Dynamics of the interaction of M2 and TRAPPC6AΔ in wt and mutant WSN virus-infected cells. A549 cells were infected with wt influenza virus WSN (A), or one of the M2 deletion mutants WSN M2Del1 (B) and WSN M2Del2 (C), at an MOI of 5. At 4, 6, 8, 10, and 14 h p.i., the infected cells were fixed and stained with mouse anti-M2 MAb 14C2 and rabbit anti-TRAPPC6A polyclonal antibody, followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Mutagenesis, Infection, Staining, Incubation

    Mutation at position 96 of M2 affects its interaction with TRAPPC6A. (A) Sequence analysis of IAV M2 at position 96. All of the IAV M2 sequences deposited in GenBank by 6 July 2014 were downloaded. The identity of the amino acids at position 96 was statistically analyzed. (B) Plasmids expressing TRAPPC6A-Myc and Flag-WSNM2 or Flag-WSNM2 with different mutations at position 96 were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Mutation at position 96 of M2 affects its interaction with TRAPPC6A. (A) Sequence analysis of IAV M2 at position 96. All of the IAV M2 sequences deposited in GenBank by 6 July 2014 were downloaded. The identity of the amino acids at position 96 was statistically analyzed. (B) Plasmids expressing TRAPPC6A-Myc and Flag-WSNM2 or Flag-WSNM2 with different mutations at position 96 were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Mutagenesis, Sequencing, Expressing, Immunoprecipitation, Western Blot

    Effect of modulation of TRAPPC6AΔ expression on the cell surface expression of viral and cellular proteins. (A) A549 cells were transfected with siRNA targeting TRAPPC6AΔ or with nontargeting siRNA for 48 h and were then infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (B) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. Cells were fixed at 8 and 10 h p.i., left nonpermeabilized, and stained with the mouse anti-M2 MAb and Alexa Fluor 488-conjugated donkey anti-mouse IgG(H+L) for M2 surface expression analysis by flow cytometry. The graph shows the fluorescence intensity of M2 surface expression. (C) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (D) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B. (E) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of HA was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-HA polyclonal antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (F) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of FGF2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-FGF2 MAb and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (G) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. At 2 h p.i., the culture medium was replaced with medium supplemented with 25 μM amantadine. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Effect of modulation of TRAPPC6AΔ expression on the cell surface expression of viral and cellular proteins. (A) A549 cells were transfected with siRNA targeting TRAPPC6AΔ or with nontargeting siRNA for 48 h and were then infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (B) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. Cells were fixed at 8 and 10 h p.i., left nonpermeabilized, and stained with the mouse anti-M2 MAb and Alexa Fluor 488-conjugated donkey anti-mouse IgG(H+L) for M2 surface expression analysis by flow cytometry. The graph shows the fluorescence intensity of M2 surface expression. (C) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (D) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B. (E) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of HA was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-HA polyclonal antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (F) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of FGF2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-FGF2 MAb and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (G) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. At 2 h p.i., the culture medium was replaced with medium supplemented with 25 μM amantadine. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Expressing, Transfection, Infection, Western Blot, Staining, Flow Cytometry, Cytometry, Fluorescence, Transduction

    TRAPPC6AΔ positively modulates influenza virus infection. (A) Endogenous expression of TRAPPC6AΔ in A549 cells. Whole lysates of A549 cells grown in 12-well plates were subjected to Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. HEK293T cell lysates transiently transfected with pCAGGS-TRAPPC6A or pCAGGS-TRAPPC6AΔ were used as a control. (B) siRNA knockdown of TRAPPC6AΔ in A549 cells. A549 cells were transfected with siRNA targeting TRAPPC6AΔ or nontargeting siRNA for 48 h. Whole-cell lysates were then collected and analyzed by Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. (C) Cell viability of siRNA-treated A549 cells measured by using the CellTiter-Glo assay. A549 cells were transfected with siRNA as described above for panel B. The data are presented as means ± standard deviations for triplicate transfections. (D) Virus replication in siRNA-treated A549 cells. Cells transfected with siRNA as described above for panel B were infected with WSN virus. At 24 and 48 h p.i., supernatants were collected and titrated for infectious virus by plaque assays in MDCK cells. Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: TRAPPC6AΔ positively modulates influenza virus infection. (A) Endogenous expression of TRAPPC6AΔ in A549 cells. Whole lysates of A549 cells grown in 12-well plates were subjected to Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. HEK293T cell lysates transiently transfected with pCAGGS-TRAPPC6A or pCAGGS-TRAPPC6AΔ were used as a control. (B) siRNA knockdown of TRAPPC6AΔ in A549 cells. A549 cells were transfected with siRNA targeting TRAPPC6AΔ or nontargeting siRNA for 48 h. Whole-cell lysates were then collected and analyzed by Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. (C) Cell viability of siRNA-treated A549 cells measured by using the CellTiter-Glo assay. A549 cells were transfected with siRNA as described above for panel B. The data are presented as means ± standard deviations for triplicate transfections. (D) Virus replication in siRNA-treated A549 cells. Cells transfected with siRNA as described above for panel B were infected with WSN virus. At 24 and 48 h p.i., supernatants were collected and titrated for infectious virus by plaque assays in MDCK cells. Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Infection, Expressing, Western Blot, Transfection, Glo Assay

    Confocal microscopy of WSN virus-infected cells stained for the Golgi apparatus or lysosomes. A549 cells were infected with the wt WSN virus at an MOI of 5. At the indicated time points, infected cells were fixed and stained with mouse anti-Giantin MAb and rabbit anti-M2 polyclonal antibody (A), mouse anti-LAMP1 MAb and rabbit anti-M2 polyclonal antibody (B), or mouse anti-LAMP1 MAb and rabbit anti-TRAPPC6A polyclonal antibody (C), followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Confocal microscopy of WSN virus-infected cells stained for the Golgi apparatus or lysosomes. A549 cells were infected with the wt WSN virus at an MOI of 5. At the indicated time points, infected cells were fixed and stained with mouse anti-Giantin MAb and rabbit anti-M2 polyclonal antibody (A), mouse anti-LAMP1 MAb and rabbit anti-M2 polyclonal antibody (B), or mouse anti-LAMP1 MAb and rabbit anti-TRAPPC6A polyclonal antibody (C), followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Confocal Microscopy, Infection, Staining, Incubation

    Effect of λ-CGN on influenza A infection in vivo . BALB/c mice were mock-infected (black) or intranasally infected with maPR8 at 5 MLD50 (red). As test groups, the virus was preincubated at room temperature for 30 min with λ-CGN at a lower dose (1 mg/kg/d, bright green) or a higher dose (5 mg/kg/d, dark green), followed by intranasal administration. Control mice received oral OSV-P twice a day (10 mg/kg/d) at 8-h intervals, starting at 4 h before viral infection (blue). Body weight (A) and mortality (B) of mice were measured every day from Days 0 to 14 post-infection. Data are expressed as the mean ± SEM from five mice

    Journal: bioRxiv

    Article Title: Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro

    doi: 10.1101/2020.08.23.255364

    Figure Lengend Snippet: Effect of λ-CGN on influenza A infection in vivo . BALB/c mice were mock-infected (black) or intranasally infected with maPR8 at 5 MLD50 (red). As test groups, the virus was preincubated at room temperature for 30 min with λ-CGN at a lower dose (1 mg/kg/d, bright green) or a higher dose (5 mg/kg/d, dark green), followed by intranasal administration. Control mice received oral OSV-P twice a day (10 mg/kg/d) at 8-h intervals, starting at 4 h before viral infection (blue). Body weight (A) and mortality (B) of mice were measured every day from Days 0 to 14 post-infection. Data are expressed as the mean ± SEM from five mice

    Article Snippet: Viral NP and HA proteins were detected using mouse anti-NP (catalog no. 11675-MM03; Sino Biological, Beijing, China) and rabbit anti-HA2 (catalog no. 86001-RM01; Sino Biological) antibodies, respectively, according to our previous report .

    Techniques: Infection, In Vivo, Mouse Assay

    Effect of λ-CGN on the intracellular entry of influenza A virus. MDCK cells were infected with PR8 (MOI, 5) and subsequently mock-treated or treated either with λ-CGN or with p-KG03 at a concentration of 10 μg/ml. At 4 h p.i. in the absence of CHX (A) or at 2.5 h in the presence of 10 μg/ml CHX (B), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×.

    Journal: bioRxiv

    Article Title: Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro

    doi: 10.1101/2020.08.23.255364

    Figure Lengend Snippet: Effect of λ-CGN on the intracellular entry of influenza A virus. MDCK cells were infected with PR8 (MOI, 5) and subsequently mock-treated or treated either with λ-CGN or with p-KG03 at a concentration of 10 μg/ml. At 4 h p.i. in the absence of CHX (A) or at 2.5 h in the presence of 10 μg/ml CHX (B), viral NP was detected with an anti-NP antibody and an Alex Fluor 488-conjugated goat anti-mouse secondary antibody (green). Cell nuclei were counterstained with DAPI (blue). Original magnification, 400×.

    Article Snippet: Viral NP and HA proteins were detected using mouse anti-NP (catalog no. 11675-MM03; Sino Biological, Beijing, China) and rabbit anti-HA2 (catalog no. 86001-RM01; Sino Biological) antibodies, respectively, according to our previous report .

    Techniques: Infection, Concentration Assay

    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Mouse Assay, Injection, Staining

    Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Mouse Assay, Neutralization

    Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Transfection, Positive Control

    Validated miRNA targets are involved in immune response and cell proliferation in the lungs. Expression levels of (A) KRAS , BLIMP1 , and CDK6 (targets of let-7f); (B) SIRT1 , ZAP70 , and MYC (targets of miR-34c); (C) CAMTA1 (target of miR-129); and (D) PTEN (target of miR-18b) in BAL from rhesus macaques (five aged and three young adults) following MEX4108 infection are shown. Expression of mRNA was normalized to expression of RPL32 . Changes in gene expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. (A–D) *let-7f, miR-34c, miR-129, miR-18-b, KRAS , SIRT1 , CAMTA1 , and PTEN ; ‡ KRAS and ZAP70 ; † CDK6 and MYC . * p

    Journal: Viral Immunology

    Article Title: microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques

    doi: 10.1089/vim.2015.0074

    Figure Lengend Snippet: Validated miRNA targets are involved in immune response and cell proliferation in the lungs. Expression levels of (A) KRAS , BLIMP1 , and CDK6 (targets of let-7f); (B) SIRT1 , ZAP70 , and MYC (targets of miR-34c); (C) CAMTA1 (target of miR-129); and (D) PTEN (target of miR-18b) in BAL from rhesus macaques (five aged and three young adults) following MEX4108 infection are shown. Expression of mRNA was normalized to expression of RPL32 . Changes in gene expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. (A–D) *let-7f, miR-34c, miR-129, miR-18-b, KRAS , SIRT1 , CAMTA1 , and PTEN ; ‡ KRAS and ZAP70 ; † CDK6 and MYC . * p

    Article Snippet: IgG and IgA binding antibody titers were measured in plasma and BAL supernatant by enzyme-linked immunosorbent assay (ELISA) using plates coated with 1 μg/mL recombinant MEX4108 HA protein (plasma) overnight at 4°C (Sino Biological, Inc., Beijing, China).

    Techniques: Expressing, Infection

    Differentially expressed miRNAs in PBMCs following MEX4108. Expression levels of miR-18b (A) , miR-20a (B) , miR-192 (C) , miR-451 (D) , miR-138 (E) , miR-193b (F) , miR-132 (G) , let-7f (H) , and miR-146b (I) in PBMCs from rhesus macaques ( n = 11; five aged and six young adults) following MEX4108 infection were determined using qRT-PCR. Expression of miRNAs was normalized to expression of U6 snRNA. Changes in microRNA expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. * p

    Journal: Viral Immunology

    Article Title: microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques

    doi: 10.1089/vim.2015.0074

    Figure Lengend Snippet: Differentially expressed miRNAs in PBMCs following MEX4108. Expression levels of miR-18b (A) , miR-20a (B) , miR-192 (C) , miR-451 (D) , miR-138 (E) , miR-193b (F) , miR-132 (G) , let-7f (H) , and miR-146b (I) in PBMCs from rhesus macaques ( n = 11; five aged and six young adults) following MEX4108 infection were determined using qRT-PCR. Expression of miRNAs was normalized to expression of U6 snRNA. Changes in microRNA expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. * p

    Article Snippet: IgG and IgA binding antibody titers were measured in plasma and BAL supernatant by enzyme-linked immunosorbent assay (ELISA) using plates coated with 1 μg/mL recombinant MEX4108 HA protein (plasma) overnight at 4°C (Sino Biological, Inc., Beijing, China).

    Techniques: Expressing, Infection, Quantitative RT-PCR

    MEX4108 infection results in increased frequency of pDCs and robust production of cytokine, chemokine, and growth factor levels in the lungs. (A) The frequencies of DCs (lin − CD14 − HLA-DR + ) and macrophages/monocytes (lin − CD14 + HLA-DR − ) in BAL cells were measured by flow cytometry. (B) The frequencies of mDC (CD123 − CD11c + ) and pDC (CD123 + CD11c − ) in BAL cells were measured by flow cytometry. Longitudinal analyses of the frequency of immune cells within BAL were carried out using a one-way repeated-measures ANOVA model, followed by Dunnett's multiple comparison post-test to explore differences between days postinfection and baseline (day 0) values. Mean ± SEM are shown. * p

    Journal: Viral Immunology

    Article Title: microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques

    doi: 10.1089/vim.2015.0074

    Figure Lengend Snippet: MEX4108 infection results in increased frequency of pDCs and robust production of cytokine, chemokine, and growth factor levels in the lungs. (A) The frequencies of DCs (lin − CD14 − HLA-DR + ) and macrophages/monocytes (lin − CD14 + HLA-DR − ) in BAL cells were measured by flow cytometry. (B) The frequencies of mDC (CD123 − CD11c + ) and pDC (CD123 + CD11c − ) in BAL cells were measured by flow cytometry. Longitudinal analyses of the frequency of immune cells within BAL were carried out using a one-way repeated-measures ANOVA model, followed by Dunnett's multiple comparison post-test to explore differences between days postinfection and baseline (day 0) values. Mean ± SEM are shown. * p

    Article Snippet: IgG and IgA binding antibody titers were measured in plasma and BAL supernatant by enzyme-linked immunosorbent assay (ELISA) using plates coated with 1 μg/mL recombinant MEX4108 HA protein (plasma) overnight at 4°C (Sino Biological, Inc., Beijing, China).

    Techniques: Infection, Flow Cytometry

    Model of differentially expressed microRNAs, gene targets, and immune response in the lungs following MEX4108 infection.

    Journal: Viral Immunology

    Article Title: microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques

    doi: 10.1089/vim.2015.0074

    Figure Lengend Snippet: Model of differentially expressed microRNAs, gene targets, and immune response in the lungs following MEX4108 infection.

    Article Snippet: IgG and IgA binding antibody titers were measured in plasma and BAL supernatant by enzyme-linked immunosorbent assay (ELISA) using plates coated with 1 μg/mL recombinant MEX4108 HA protein (plasma) overnight at 4°C (Sino Biological, Inc., Beijing, China).

    Techniques: Infection

    Pandemic H1N1 virus replicates to similar levels in young and aged macaques. Viral loads were measured using qRT-PCR using primers and probes specific for MEX4108 hemagglutinin (HA) in throat swabs (A) , nasal swabs (B) , ocular swabs (C) , and BAL fluid (D) . Viral genome copy number data were log transformed with base 10 and longitudinal changes of viral genome copy number between aged and young adults were compared using a two-way ANOVA, followed by Bonferroni's multiple comparison post-test to determine differences in viral load. Longitudinal changes were compared using repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test to explore differences between days postinfection and baseline (day 0) values, mean ± SEM are shown. (A–D) * for aged animals; ‡ for young adult animals; *** ,‡‡ p

    Journal: Viral Immunology

    Article Title: microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques

    doi: 10.1089/vim.2015.0074

    Figure Lengend Snippet: Pandemic H1N1 virus replicates to similar levels in young and aged macaques. Viral loads were measured using qRT-PCR using primers and probes specific for MEX4108 hemagglutinin (HA) in throat swabs (A) , nasal swabs (B) , ocular swabs (C) , and BAL fluid (D) . Viral genome copy number data were log transformed with base 10 and longitudinal changes of viral genome copy number between aged and young adults were compared using a two-way ANOVA, followed by Bonferroni's multiple comparison post-test to determine differences in viral load. Longitudinal changes were compared using repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test to explore differences between days postinfection and baseline (day 0) values, mean ± SEM are shown. (A–D) * for aged animals; ‡ for young adult animals; *** ,‡‡ p

    Article Snippet: IgG and IgA binding antibody titers were measured in plasma and BAL supernatant by enzyme-linked immunosorbent assay (ELISA) using plates coated with 1 μg/mL recombinant MEX4108 HA protein (plasma) overnight at 4°C (Sino Biological, Inc., Beijing, China).

    Techniques: Quantitative RT-PCR, Transformation Assay

    Differentially expressed miRNAs in BAL cells following MEX4108. Expression levels of let-7f (A) , miR-34c (B) , miR-129 (C) , miR-18b (D) , miR-146b (E) , miR-132 (F) , miR-192 (G) , and miR-138 (H) in BAL cells from rhesus macaques ( n = 8; five aged and three young adults) following MEX4108 infection were determined using qRT-PCR. Expression of miRNAs was normalized to expression of U6 snRNA. Changes in microRNA expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. * p

    Journal: Viral Immunology

    Article Title: microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques

    doi: 10.1089/vim.2015.0074

    Figure Lengend Snippet: Differentially expressed miRNAs in BAL cells following MEX4108. Expression levels of let-7f (A) , miR-34c (B) , miR-129 (C) , miR-18b (D) , miR-146b (E) , miR-132 (F) , miR-192 (G) , and miR-138 (H) in BAL cells from rhesus macaques ( n = 8; five aged and three young adults) following MEX4108 infection were determined using qRT-PCR. Expression of miRNAs was normalized to expression of U6 snRNA. Changes in microRNA expression postinfection were determined using one-way repeated-measures ANOVA, followed by Dunnett's multiple comparison post-test. Mean ± SEM are shown. * p

    Article Snippet: IgG and IgA binding antibody titers were measured in plasma and BAL supernatant by enzyme-linked immunosorbent assay (ELISA) using plates coated with 1 μg/mL recombinant MEX4108 HA protein (plasma) overnight at 4°C (Sino Biological, Inc., Beijing, China).

    Techniques: Expressing, Infection, Quantitative RT-PCR