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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
Rabbit Anti Nep, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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anti-influenza a ns2 (nep) rabbit polyclonal  (Thermo Fisher)
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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rabbit anti- neprilysin (nep) antibody  (Millipore)
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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rabbit polyclonal anti-influenza virus ns2  (GeneTex)
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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rabbit polyclonal anti-ns2 a01499  (GenScript corporation)
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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rabbit anti-ns2 antibody  (GeneTex)
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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polyclonal rabbit anti-ns2  (Thermo Fisher)
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a Schematic representation of the experimental design for analyzing <t>H9N2-NS2</t> interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.
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a Schematic representation of the experimental design for analyzing H9N2-NS2 interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Schematic representation of the experimental design for analyzing H9N2-NS2 interacting proteins via LC-MS/MS proteomics. b HEK293T cells were transfected with Flag-H9N2-NS2 or an empty vector (Vec) control. After 24 hours, the cells were infected with H9N2 virus at a multiplicity of infection (MOI) of 0.01. Twenty-four hours post-infection, cells were harvested for immunoprecipitation (IP) using anti-Flag beads. Protein complexes were eluted with 3× Flag peptides and analyzed by western blotting (WB) (left) or LC-MS/MS (right), with n = 3 independent biological replicates. c Co-IP experiments showing the interaction of Flag-H9N2-NS2 with endogenous huANP32A/B in HEK293T cells. d Co-IP experiments showing the interaction of H9N2-NS2 with exogenous huANP32A/B in HEK293T-TKO cells stably expressing huANP32A-Flag or huANP32B-Flag. e Direct interaction between NS2 from H9N2 infection and huANP32A/B was measured using a proximity ligation assay (PLA) in HEK293T cells, with controls using one antibody. The PLA was performed 24 hours post-infection. f Schematic representation of the BiFC fusion proteins. g Confocal experiments using a BiFC assay showing the interaction of NS2 with huANP32A/B in HEK293T cells. h Co-IP experiments showing that mutations (V109G and E110G, VE/GG) in NS2-SIM suppressed its interaction with huANP32A/B in HEK293T-TKO cells that stably express huANP32A-Flag or huANP32B-Flag. i Confocal experiments using a BiFC assay showing that VE/GG mutations in NS2-SIM suppress its interaction with huANP32A or huANP32B. j , k Flow cytometry analysis using a BiFC assay showing that NS2 interacts with huANP32A ( j ) or huANP32B ( k ) depending on its SIM. Plasmids were transfected individually or in pairs into HEK293T cells. After 24 hours, the mean fluorescence intensity (MFI) of BiFC signals was measured by flow cytometry and are presented as relative values to the signal from untransfected cells. Western blots showing the protein expression of the indicated plasmids in HEK293T cells. Error bars represent means ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-tailed unpaired t-test . Experiments in ( c ), ( d ), and ( h ) were repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Control, Infection, Virus, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Proximity Ligation Assay, Bimolecular Fluorescence Complementation Assay, Flow Cytometry, Fluorescence, Two Tailed Test

a Ni 2+ -NTA bead affinity pull-down assay results showing that SUMOylation of huANP32A or huANP32B in HEK293T cells was enhanced by overexpression of Ubc9. b , c Co-IP experiments showing that overexpression of Ubc9 promoted the interaction between H9N2-NS2 and huANP32A ( b ) or huANP32B ( c ). d Ni 2+ -NTA bead affinity pull-down assay results showing that SUMOylation of huANP32A or huANP32B in HEK293T cells was reduced by overexpression of SENP1. e Co-IP experiments showing overexpression of SENP1 suppressed the interaction between H9N2-NS2 and huANP32A or huANP32B. f Ni 2+ -NTA bead affinity pull-down assay results showing the SUMOylation of huANP32A in HEK293T cells was reduced by treatment with TAK-981. g Co-IP experiments showing that treatment with TAK-981 suppressed NS2-huANP32A interaction. h Illustration of the interaction model between NS2 and huANP32A/B mediated by the SIM-SUMO working pattern. All Western blot experiments were independently repeated at least twice with consistent results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Ni 2+ -NTA bead affinity pull-down assay results showing that SUMOylation of huANP32A or huANP32B in HEK293T cells was enhanced by overexpression of Ubc9. b , c Co-IP experiments showing that overexpression of Ubc9 promoted the interaction between H9N2-NS2 and huANP32A ( b ) or huANP32B ( c ). d Ni 2+ -NTA bead affinity pull-down assay results showing that SUMOylation of huANP32A or huANP32B in HEK293T cells was reduced by overexpression of SENP1. e Co-IP experiments showing overexpression of SENP1 suppressed the interaction between H9N2-NS2 and huANP32A or huANP32B. f Ni 2+ -NTA bead affinity pull-down assay results showing the SUMOylation of huANP32A in HEK293T cells was reduced by treatment with TAK-981. g Co-IP experiments showing that treatment with TAK-981 suppressed NS2-huANP32A interaction. h Illustration of the interaction model between NS2 and huANP32A/B mediated by the SIM-SUMO working pattern. All Western blot experiments were independently repeated at least twice with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Pull Down Assay, Over Expression, Co-Immunoprecipitation Assay, Western Blot

a Schematic illustrating two strategies employed for the investigation of whether the SIM-SUMO-mediated interactions between NS2 and huANP32A/B are required for NS2-SIM to promote huANP32A/B-supported AIV vPol activity. b , c Minigenome assays in HEK293T-TKO cells showing that disruption of the H9N2 or H7N9 NS2-SIM integrity impairs the ability of NS2 to enhance huANP32A/B-supported H9N2 ( b ) or H7N9 (PB2-627E) ( c ) vPol activity, respectively. d Schematic model of the generation of lysine-free mutant of huANP32A/B (huANP32A/B-K0). e Ni 2+ -NTA bead affinity pull-down assay results showing that huANP32A/B-K0 could not be SUMOylated in HEK293T cells. f , g Minigenome assays in HEK293T-TKO cells showing that the ability of NS2 to promote huANP32A-K0 ( f ) or huANP32B-K0 ( g )-supported H9N2 or H7N9 (PB2-627E) vPol activity was greatly reduced. The accompanying western blots show the expression of ANP32A-Flag/ANP32B-HA constructs and vRNP components (PA/NP). h Replication kinetics of avian H9N2 virus. MDCK-TKO cells or those tranfected with huANP32A-HA or huANP32A-K0-HA were infected (MOI = 0.01), and viral titers were determined at the indicated time points. i Western blots showing equal expression of HA-tagged ANP32A in transfected MDCK-TKO cells. j The replication kinetics of avian H9N2 virus were assessed in MDCK-TKO cells transfected with either empty vector, huANP32B-HA or huANP32B-K0-HA (MOI = 0.01). Viral titers were determined at the indicated time points. k Western blots showing equal expression of HA-tagged ANP32B in transfected MDCK-TKO cells. In ( b ), ( c ), ( f to h ) and ( j ), error bars represent mean ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-way ANOVA ( b and c ) or two-tailed unpaired t-test ( f and g ). Experiments in ( e ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Schematic illustrating two strategies employed for the investigation of whether the SIM-SUMO-mediated interactions between NS2 and huANP32A/B are required for NS2-SIM to promote huANP32A/B-supported AIV vPol activity. b , c Minigenome assays in HEK293T-TKO cells showing that disruption of the H9N2 or H7N9 NS2-SIM integrity impairs the ability of NS2 to enhance huANP32A/B-supported H9N2 ( b ) or H7N9 (PB2-627E) ( c ) vPol activity, respectively. d Schematic model of the generation of lysine-free mutant of huANP32A/B (huANP32A/B-K0). e Ni 2+ -NTA bead affinity pull-down assay results showing that huANP32A/B-K0 could not be SUMOylated in HEK293T cells. f , g Minigenome assays in HEK293T-TKO cells showing that the ability of NS2 to promote huANP32A-K0 ( f ) or huANP32B-K0 ( g )-supported H9N2 or H7N9 (PB2-627E) vPol activity was greatly reduced. The accompanying western blots show the expression of ANP32A-Flag/ANP32B-HA constructs and vRNP components (PA/NP). h Replication kinetics of avian H9N2 virus. MDCK-TKO cells or those tranfected with huANP32A-HA or huANP32A-K0-HA were infected (MOI = 0.01), and viral titers were determined at the indicated time points. i Western blots showing equal expression of HA-tagged ANP32A in transfected MDCK-TKO cells. j The replication kinetics of avian H9N2 virus were assessed in MDCK-TKO cells transfected with either empty vector, huANP32B-HA or huANP32B-K0-HA (MOI = 0.01). Viral titers were determined at the indicated time points. k Western blots showing equal expression of HA-tagged ANP32B in transfected MDCK-TKO cells. In ( b ), ( c ), ( f to h ) and ( j ), error bars represent mean ± SD from n = 3 independent biological replicates; Statistical significance was determined by two-way ANOVA ( b and c ) or two-tailed unpaired t-test ( f and g ). Experiments in ( e ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Activity Assay, Disruption, Mutagenesis, Pull Down Assay, Western Blot, Expressing, Construct, Virus, Infection, Transfection, Plasmid Preparation, Two Tailed Test

a Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated constructs on H9N2 vPol activity. Values above K0 were statistically analyzed using one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32A-K0 (error bars represent the mean ± SD of n = 4 independent biological replicates). b Schematic representation of the huANP32A-K0 mutants generated. Western blots demonstrate comparable expression levels for all indicated constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of MDCK-TKO cells stably reconstituted with the indicated huANP32A-K0-Flag constructs or empty vector. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K153 sites of huANP32A can be modified by SUMO1. h Co-IP experiments showing that the K68R/K153R mutations in huANP32A suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32A. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-Flag or huANP32A-K68R/K153R-Flag (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32A-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). In ( g , h ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated constructs on H9N2 vPol activity. Values above K0 were statistically analyzed using one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32A-K0 (error bars represent the mean ± SD of n = 4 independent biological replicates). b Schematic representation of the huANP32A-K0 mutants generated. Western blots demonstrate comparable expression levels for all indicated constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of MDCK-TKO cells stably reconstituted with the indicated huANP32A-K0-Flag constructs or empty vector. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K153 sites of huANP32A can be modified by SUMO1. h Co-IP experiments showing that the K68R/K153R mutations in huANP32A suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32A. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-Flag or huANP32A-K68R/K153R-Flag (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32A-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). In ( g , h ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Plasmid Preparation, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test

a Minigenome assays in HEK293T-TKO cells comparing the effect of indicated huANP32B-K0 constructs on the H9N2 vPol activity. Values higher than K0 were analyzed via one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32B-K0 (error bars represent the mean ± SD of n = 3 independent biological replicates). b Schematic representation of the huANP32B-K0 mutants generated. Western blots confirmed comparable expression levels of all constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-K0-Flag constructs. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K116 sites of huANP32B can be modified by SUMO1. h Co-IP experiments showing that the K68R/K116R mutations in huANP32B suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32B. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). Experiments in ( g and h ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of indicated huANP32B-K0 constructs on the H9N2 vPol activity. Values higher than K0 were analyzed via one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32B-K0 (error bars represent the mean ± SD of n = 3 independent biological replicates). b Schematic representation of the huANP32B-K0 mutants generated. Western blots confirmed comparable expression levels of all constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-K0-Flag constructs. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K116 sites of huANP32B can be modified by SUMO1. h Co-IP experiments showing that the K68R/K116R mutations in huANP32B suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32B. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). Experiments in ( g and h ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test

a NS2 failed to promote the binding of huANP32A-K0 to H9N2 vRNP. HEK293T-TKO cells were transfected with expression vectors for the indicated huANP32A-Flag constructs (0.4 μg), together with H9N2-PB1 (0.4 μg), H9N2-PB2 (0.4 μg), H9N2-PA (0.2 μg), H9N2-NP (0.8 μg), and vRNA luciferase reporter (0.4 μg) and either with or without H9N2-NS2 (50 ng). The transfected cells were collected for IP and western blot analysis 24 hours post-transfection. b NS2 promotes huANP32A-K0-R68K/R153K binding to H9N2 vRNP. c Effect of K68R, K153R or K68R/K153R mutations in huANP32A on its interaction with H9N2 vRNP in the presence of NS2. d NS2 did not enhance huANP32B-K0 binding to H9N2 vRNP. e NS2 promotes huANP32B-K0-R68K/R116K binding to H9N2 vRNP. f Effect of K68R, K116R or K68R/K116R mutations in huANP32B on its interaction with H9N2 vRNP in the presence of NS2. Experiments in (b to f) were performed as in Fig. 8a. g Effect of NS2 on the H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32A or huANP32A-K0. HEK293T-TKO cells were transfected with different ANP32A-V5 (0.4 μg), H9N2-NP-Flag (0.8 μg) and polymerase plasmids from H9N2 (0.2 μg PA, 0.4 μg PB1, and 0.4 μg PB2) together with vRNA luciferase reporter (0.4 μg) and H9N2-NS2 (50 ng). After anti-Flag immunoprecipitation at 24 hours post-transfection, the indicated proteins were analyzed with western blotting. h Effect of NS2 on the H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32A-K0 or huANP32A-K0-R68K/R153K. i Measurement of H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with huANP32A or the indicated mutants. j Effect of NS2 on H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32B or huANP32B-K0. k Effect of NS2 on H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32B-K0 or huANP32B-K0-R68K/R116K. l Measurement of H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with huANP32B or the mutants indicated. Experiments in ( h to l ) were performed as in Fig. 8g. Experiments in ( a to l ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: a NS2 failed to promote the binding of huANP32A-K0 to H9N2 vRNP. HEK293T-TKO cells were transfected with expression vectors for the indicated huANP32A-Flag constructs (0.4 μg), together with H9N2-PB1 (0.4 μg), H9N2-PB2 (0.4 μg), H9N2-PA (0.2 μg), H9N2-NP (0.8 μg), and vRNA luciferase reporter (0.4 μg) and either with or without H9N2-NS2 (50 ng). The transfected cells were collected for IP and western blot analysis 24 hours post-transfection. b NS2 promotes huANP32A-K0-R68K/R153K binding to H9N2 vRNP. c Effect of K68R, K153R or K68R/K153R mutations in huANP32A on its interaction with H9N2 vRNP in the presence of NS2. d NS2 did not enhance huANP32B-K0 binding to H9N2 vRNP. e NS2 promotes huANP32B-K0-R68K/R116K binding to H9N2 vRNP. f Effect of K68R, K116R or K68R/K116R mutations in huANP32B on its interaction with H9N2 vRNP in the presence of NS2. Experiments in (b to f) were performed as in Fig. 8a. g Effect of NS2 on the H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32A or huANP32A-K0. HEK293T-TKO cells were transfected with different ANP32A-V5 (0.4 μg), H9N2-NP-Flag (0.8 μg) and polymerase plasmids from H9N2 (0.2 μg PA, 0.4 μg PB1, and 0.4 μg PB2) together with vRNA luciferase reporter (0.4 μg) and H9N2-NS2 (50 ng). After anti-Flag immunoprecipitation at 24 hours post-transfection, the indicated proteins were analyzed with western blotting. h Effect of NS2 on the H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32A-K0 or huANP32A-K0-R68K/R153K. i Measurement of H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with huANP32A or the indicated mutants. j Effect of NS2 on H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32B or huANP32B-K0. k Effect of NS2 on H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with either huANP32B-K0 or huANP32B-K0-R68K/R116K. l Measurement of H9N2 vRNP assembly in HEK293T-TKO cells reconstituted with huANP32B or the mutants indicated. Experiments in ( h to l ) were performed as in Fig. 8g. Experiments in ( a to l ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Binding Assay, Transfection, Expressing, Construct, Luciferase, Western Blot, Immunoprecipitation

A Both huANP32A and huANP32B are SUMOylated by the E3 SUMO ligase PIAS2α and deSUMOylated by SENP1. SUMOylation of huANP32A and huANP32B increases upon AIV infection. B huANP32A and huANP32B cannot efficiently support AIV vPol activity due to weak interactions between AIV vRNP and huANP32A/B, as well as inefficient AIV vRNP assembly. C During AIV infection, the NS2 protein is recruited to the replication platform by SUMOylated huANP32A/B via the SIM-SUMO interaction pattern. The recruited NS2 uses its SIM to mediate an intimate association with K68/K153-SUMO in huANP32A or K68/K116-SUMO in huANP32B, which promotes huANP32A/B-supported AIV vPol activity by overcoming the defects in AIV vRNP-huANP32A/B interactions and AIV vRNP assembly. This figure was created with BioRender ( https://www.biorender.com ).

Journal: Nature Communications

Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

doi: 10.1038/s41467-024-55034-y

Figure Lengend Snippet: A Both huANP32A and huANP32B are SUMOylated by the E3 SUMO ligase PIAS2α and deSUMOylated by SENP1. SUMOylation of huANP32A and huANP32B increases upon AIV infection. B huANP32A and huANP32B cannot efficiently support AIV vPol activity due to weak interactions between AIV vRNP and huANP32A/B, as well as inefficient AIV vRNP assembly. C During AIV infection, the NS2 protein is recruited to the replication platform by SUMOylated huANP32A/B via the SIM-SUMO interaction pattern. The recruited NS2 uses its SIM to mediate an intimate association with K68/K153-SUMO in huANP32A or K68/K116-SUMO in huANP32B, which promotes huANP32A/B-supported AIV vPol activity by overcoming the defects in AIV vRNP-huANP32A/B interactions and AIV vRNP assembly. This figure was created with BioRender ( https://www.biorender.com ).

Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

Techniques: Infection, Activity Assay