a new caledonia 20 1999  (Sino Biological)


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    Name:
    Influenza A H1N1 A New Caledonia 20 1999 Hemagglutinin HA Protein His Tag
    Description:
    A DNA sequence encoding the influenza hemagglutinin A New Caledonia 20 1999 H1N1 Met 1 Gln 528 AAP34324 1 HA1 HA2 uncleaved was fused with a C terminal polyhistidine tag
    Catalog Number:
    11683-V08H
    Price:
    None
    Category:
    recombinant protein
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological a new caledonia 20 1999
    Hemagglutination inhibition (HI) assay. Serum HI titers against vaccine influenza virus strains A/South Australia/55/2014 (H3N2) (A), B/Phuket/3073/2013 (B), and A/California/7/2009 (H1N1) (C) and a nonvaccine strain, A/New <t>Caledonia/20/1999</t> (H1N1) (D).
    A DNA sequence encoding the influenza hemagglutinin A New Caledonia 20 1999 H1N1 Met 1 Gln 528 AAP34324 1 HA1 HA2 uncleaved was fused with a C terminal polyhistidine tag
    https://www.bioz.com/result/a new caledonia 20 1999/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine"

    Article Title: Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine

    Journal: Journal of Virology

    doi: 10.1128/JVI.00285-16

    Hemagglutination inhibition (HI) assay. Serum HI titers against vaccine influenza virus strains A/South Australia/55/2014 (H3N2) (A), B/Phuket/3073/2013 (B), and A/California/7/2009 (H1N1) (C) and a nonvaccine strain, A/New Caledonia/20/1999 (H1N1) (D).
    Figure Legend Snippet: Hemagglutination inhibition (HI) assay. Serum HI titers against vaccine influenza virus strains A/South Australia/55/2014 (H3N2) (A), B/Phuket/3073/2013 (B), and A/California/7/2009 (H1N1) (C) and a nonvaccine strain, A/New Caledonia/20/1999 (H1N1) (D).

    Techniques Used: HI Assay

    Related Articles

    Recombinant:

    Article Title: In situ self-assembly of gold nanoparticles on hydrophilic and hydrophobic substrates for influenza virus-sensing platform
    Article Snippet: .. Recombinant influenza virus A (New Caledonia/20/1999) (H1N1) (Cat No. 11683-V08H) and influenza A (H3N2) hemagglutinin monoclonal antibodies (Anti-H3N2 antibody HA MAb, Lot: HB04N0160) were purchased from Sino Biological, Inc. (Beijing, China). ..

    Article Title: Detection of influenza virus using peroxidase-mimic of gold nanoparticles.
    Article Snippet: .. Recombinant influenza virus A (New Caledonia/20/1999) (H1N1) (Cat No.: 11683-V08H) and anti-H3(H3N2) antibody HA MAb (Lot: HB04N0160) were purchased from Sino Biological Inc. (Beijing, China). ..

    Article Title: Magnetic Nanozyme-Linked Immunosorbent Assay for Ultrasensitive Influenza A Virus Detection.
    Article Snippet: .. Rapid and sensitive detection of influenza virus is of soaring importance to prevent further spread of infections and adequate clinical treatment. ..

    Purification:

    Article Title: Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine
    Article Snippet: .. Simian immunodeficiency virus (SIV) gp120 and purified HA from A/Switzerland/9715293/2013 (H3N2), A/Perth/16/2009 (H3N2), A/California/7/2009 (H1N1), A/New Caledonia/20/1999 (H1N1), B/Phuket/3073/2013 (Yamagata), and B/Florida/4/2006 (Yamagata) were purchased from Sinobiological, Beijing, China. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Androgen supplementation improves some but not all aspects of immune senescence in aged male macaques
    Article Snippet: .. Plasma IgG antibody titers were measured on the day of vaccination and then on days 7, 14, and 28 or 35 (for MVA and H1N1 respectively) by enzyme-linked immunosorbent assay (ELISA) using plates coated with modified vaccinia virus (MVA) lysate (1 μg/mL) or with Influenza A (H1N1) 2009 Monovalent Vaccine overnight at 4 °C (0.15 μg HA antigen/mL Sino Biological, Inc., Beijing, China). ..

    Modification:

    Article Title: Androgen supplementation improves some but not all aspects of immune senescence in aged male macaques
    Article Snippet: .. Plasma IgG antibody titers were measured on the day of vaccination and then on days 7, 14, and 28 or 35 (for MVA and H1N1 respectively) by enzyme-linked immunosorbent assay (ELISA) using plates coated with modified vaccinia virus (MVA) lysate (1 μg/mL) or with Influenza A (H1N1) 2009 Monovalent Vaccine overnight at 4 °C (0.15 μg HA antigen/mL Sino Biological, Inc., Beijing, China). ..

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    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
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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
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    93
    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: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Sino Biological h1n1
    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 <t>(H1N1)</t> or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p
    H1n1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    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

    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections

    doi: 10.1016/j.omtm.2017.09.003

    Figure Lengend Snippet: Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Article Snippet: ELISA plates coated with 200 ng of H1N1 (A/WSN/33) HA, 50 ng of H3N2 (A/Aichi/2/1968) HA1 or HA subunit, or 200 ng of H10N8 (A/Jiangxi-Donghu/346/2013) HA (Sino Biologicals, Beijing, China) were blocked and incubated with serially diluted mouse serum.

    Techniques: Mouse Assay