Anti-Influenza A Virus M2 Protein antibody [14C2]

• ICC

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Immunocytochemistry - Anti-Influenza A Virus M2 Protein antibody [14C2] (AB5416)

Immunofluorescence staining of infected dog MDCK cells using ab5416.

• WB

CiteAb

Western blot - Anti-Influenza A Virus M2 Protein antibody [14C2] (AB5416)

Western Blotting using Anti-Influenza A Virus M2 Protein antibody [14C2], ab5416. Publication image from Moriyama, M. et al., 2019, Nat Commun, 31604929. Legend direct from paper.

Influenza virus stimulates DDX41-dependent IFN-β gene expression. a cGAS-293FT cells transfected with siRNA targeting DDX41 or control siRNA were infected with influenza virus for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). b, c cGAS-293FT cells were infected with WT (left panel) or δNS1 influenza virus (right panel) for 24 h in the presence or absence of LFM-A13 (100 µM) (b). WT or DDX41-deficient STING-A549 cells were infected with PR8 (left panel), or EMCV (right panel) for 24 h (c). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. d Pure cytosolic fraction prepared from digitonin extracts of mock- or δNS1 influenza virus-infected cGAS-293FT cells were treated with DNase I or RNase H. Cytosolic mtDNA was assessed by quantitative PCR. e HEK293FT cells were transfected with DNA extracted from DNase I- or RNase H-treated pure cytosolic fraction for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. f STING-A549 cells transfected with siRNA targeting DDX41 or control siRNA were infected with PR8 virus for 24 h. Cell lysates were collected at 24 h post infection and blotted using the indicated antibodies (left panel). Pure cytosolic extracts were collected at 24 h post infection and analyzed for cGAMP by ELISA (right panel). g, h HEK293FT cells were transfected with siRNA targeting DDX41. Two days later, cells were transfected with the expression plasmid encoding Flag-tagged DDX41 or DDX41 (Y414F) mutant. Twenty-four hours after DNA transfection, the cells were infected with WT (g) δNS1 influenza virus (h) for 24 h. Cell lysates were collected at 24 h post infection and blotted using the indicated antibodies (left panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). These data are from three independent experiments (mean ± s.e.m.). **P < 0.01, ***P < 0.001; n.s., not significant (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file

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• WB

CiteAb

Western blot - Anti-Influenza A Virus M2 Protein antibody [14C2] (AB5416)

Western Blotting using Anti-Influenza A Virus M2 Protein antibody [14C2], ab5416. Publication image from Moriyama, M. et al., 2019, Nat Commun, 31604929. Legend direct from paper.

Influenza virus NS1 protein associates with mtDNA. a HEK293FT cells were transfected with the expression plasmid encoding EGFP, Flag-tagged M2, NS1, or NS138/41 mutant. Twenty-four hours after transfection, the cells were infected with δNS1 influenza virus for 24 h. Pure cytosolic extracts prepared from digitonin extracts of δNS1 influenza virus-infected cells were immunoprecipitated with mouse monoclonal antibody against Flag, followed by immunoblotting of immunoprecipitates with rabbit polyclonal antibody against Flag (left panel). DNA was extracted from immunoprecipitated samples using QIAquick Nucleotide Removal kit (QIAGEN). NS1-bound mtDNA was assessed by quantitative PCR (right panel). b HEK293FT cells were transfected with the expression plasmid encoding EGFP, Flag-tagged NS1, or NS1 38/41 mutant. Twenty-four hours after transfection, the cells were infected with δNS1 influenza virus for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR (middle panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). c HEK293FT cells were infected with WT (rgPR8) or rgPR8/NS138/41A influenza virus at MOI of 1 for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). Cytosolic mtDNA (middle panel) and IFN-β mRNA levels (right panel) were assessed by quantitative PCR. d cGAS-293FT cells transfected with siRNA targeting STING or control siRNA were infected with rgPR8/NS138/41A influenza virus for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. These data are from three independent experiments (mean ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file

false

• WB

CiteAb

Western blot - Anti-Influenza A Virus M2 Protein antibody [14C2] (AB5416)

Western Blotting using Anti-Influenza A Virus M2 Protein antibody [14C2], ab5416. Publication image from Moriyama, M. et al., 2019, Nat Commun, 31604929. Legend direct from paper.

Influenza virus stimulates cGAS- and STING-dependent IFN-β gene expression in mouse lung fibroblast. a, b Primary lung fibroblast prepared from WT, cGAS-, STING-, and MAVS-deficient mice were infected with WT PR8 (a) or δNS1 influenza virus (b). IFN-β mRNA levels were assessed by quantitative PCR with GAPDH as an internal control. c Samples from HEK293FT cells stably expressing EGFP (EGFP-293FT) or cGAS (cGAS-293FT) were infected with PR8 or EMCV. Cell lysates were collected at 9 h post infection and blotted using the indicated antibodies. d EGFP-293FT or cGAS-293FT cells were infected with influenza virus (left panel) or EMCV (right panel) for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. e, f STING-A549 cells (e) or lung fibroblasts (f) were infected with PR8 virus. Pure cytosolic extracts were collected at indicated time points and analyzed for cGAMP by ELISA. These data are from three independent experiments (a, b, d–f; mean ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file

false

• WB

CiteAb

Western blot - Anti-Influenza A Virus M2 Protein antibody [14C2] (AB5416)

Western Blotting using Anti-Influenza A Virus M2 Protein antibody [14C2], ab5416. Publication image from Moriyama, M. et al., 2019, Nat Commun, 31604929. Legend direct from paper.

Ion channel activity of influenza virus M2 protein is essential for mtDNA release. a HEK293FT cells were transfected with the expression plasmid encoding EGFP or Flag-tagged influenza virus proteins. Cell lysates were collected at 24 h post transfection and analyzed by immunoblot with mouse monoclonal antibody against Flag or EGFP (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (right panel). b HEK293FT cells were transfected with the expression plasmid encoding EGFP or Flag-tagged WT or mutant M2 protein. Cell lysates were collected at 24 h post transfection and analyzed by immunoblot using indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (right panel). c HEK293FT cells were transfected with the expression plasmid encoding EGFP or Flag-tagged M2 protein in the presence or absence of BAPTA-AM (20 µM) or Mito-TEMPO (500 µM). Cell lysates were collected at 24 h post transfection and blotted using the indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (right panel). d HEK293FT cells were transfected with siRNA targeting MAVS or control siRNA. Two days later, cells were transfected with the expression plasmid encoding EGFP or Flag-tagged M2 protein. Cell lysates were collected at 24 h post DNA transfection and blotted using the indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post DNA transfection (right panel). e, f HEK293FT cells were infected with WT (rgPR8), M2del29–31 virus (rgPR8/M2del29–31) (e), or amantadine sensitive-recombinant influenza virus (rgPR8/M2N31S) in the presence or absence of amantadine (100 µM) (f). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post infection. These data are from three independent experiments (a–f; mean ± s.e.m.). ***P < 0.001; n.s., not significant (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file

false

• WB

CiteAb

Western blot - Anti-Influenza A Virus M2 Protein antibody [14C2] (AB5416)

Western Blotting using Anti-Influenza A Virus M2 Protein antibody [14C2], ab5416. Publication image from Moriyama, M. et al., 2019, Nat Commun, 31604929. Legend direct from paper.

Connexin 43 amplifies influenza virus-induced STING-dependent innate immune signaling. a Schematic representation of experimental setup (left panel). cGAS-293FT cells were infected with δNS1 influenza virus (purple). Six hours later, uninfected WT (red) or STING KO (green) HEK293FT cells were added to the δNS1 influenza virus-infected cGAS-293FT cells (purple) and co-cultured for additional 18 h. IFN-β mRNA levels were assessed by quantitative PCR at 24 h post infection (right panel). b cGAS-293FT cells infected with δNS1 influenza virus in the presence or absence of CBX (160 µM). IFN-β mRNA levels were assessed by quantitative PCR at 24 h post infection. c cGAS-293FT cells were transfected with the expression plasmid encoding HA-tagged IRF3. Twenty-four hours after transfection, the cells were infected with δNS1 influenza virus in the presence or absence of CBX (160 µM). Cell lysates were collected at 12 h post infection and blotted using the indicated antibodies. d Samples from cGAS-293FT cells transfected with siRNA targeting connexin 43 (CX43) or control siRNA were blotted using the indicated antibodies (left panel) or intracellularly stained with CX43-specific antibody and analyzed by flow cytometry (right panel). e Schematic representation of experimental setup (left panel). cGAS-293FT cells transfected with siRNA targeting connexin 43 (CX43) or control siRNA were infected with δNS1 influenza virus (purple). Six hours later, uninfected HEK293FT cells (red) were added to the δNS1 influenza virus-infected cGAS-293FT cells (purple) and co-cultured for additional 18 h. IFN-β mRNA levels were assessed by quantitative PCR at 24 h post infection (right panel). These data are from three independent experiments (a, b, e; mean ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file

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