Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: MHCII-Targeting A/B Heterodimeric and C H 3 Homodimeric DNA Vaccines Both Confer Complete Protection against Influenza Infection In (A)–(D), BALB/c mice were vaccinated with 100 μg of (two plasmids) DNA i.m./EP with the indicated A/B heterodimeric vaccines expressing HA from PR8 (H1N1). In (E) and (F), A/B heterodimeric and C H 3-based homodimeric vaccines were compared, using 100 μg (A/B heterodimer, two plasmids) and 50 μg (C H 3 homodimer, one plasmid) of DNA i.m./EP. (A, B, E, and F) Levels of HA-specific IgG1 (A and E) and IgG2a (B and F) serum antibodies 6 weeks after immunization (mean ± SEM, **p < 0.01, Mann-Whitney test; ND, not detectable). (C and G) 15 (C) or 4 (G) weeks after vaccination, the mice were infected with a lethal dose of PR8 influenza virus (7.5 × LD 50 ). Weight was followed for 10 days (mean ± SEM; *p < 0.05, **p < 0.01, Mann-Whitney test, comparing targeting to non-targeting vaccines). (D and H) Survival curves of the vaccinated mice. Non-significant (ns, p = 0.0549) or significant p values are indicated (**p < 0.01, Mantel-Cox test). n = 6/group in (A)–(D), n = 8/group in (E)–(H). Weight loss of 20% was defined as the humane endpoint, at which time point the mice were euthanized.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: Infection, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: Diverse Targeting Units and Antigenic Units Are Efficiently Expressed on Both the A and B Arm of the Heterodimeric Protein Vaccine In Vitro (A–D) Targeting units, dimerization units, and antigenic units are indicated by symbols (given in boxes). HEK293E cells were transiently transfected with combinations of various DNA vaccine plasmids, theoretically yielding the vaccine molecules depicted in the top rows. Supernatants were analyzed in sandwich ELISAs as indicated (mean + SD). Expected binding of coat mAb (green) or detection mAb (red) to protein vaccine units (squares) is indicated by colors. Positive or negative signal obtained in sandwich ELISAs are indicated by + or −. (A) The targeting units, Xcl1 chemokine and scFv αNIP , were genetically fused to either A or B chains in combination with OVA and mCherry as antigenic units, as indicated. Homodimeric scFv αNIP -C H 3-scFv 315 was included as a negative control. (B) The targeting units, Xcl1 and scFv αNIP , were genetically fused to either A or B chains in combination with antigenic units OVA and mCherry, as indicated. (C) MIP1α and scFv αNIP were expressed in all possible combinations in the targeting units of A/B heterodimers and C H 3-based homodimers, as indicated. The antigenic unit, scFv 315 , was invariable. (D) The targeting units, scFv αMHCII and scFv αNIP , were expressed with antigenic units HA from PR8 and Cal07 influenza strains in A/B heterodimers, as indicated.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: In Vitro, Transfection, Binding Assay, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: Antigen-Specific Responses Are Induced Irrespective of Whether the Antigen Is Fused to the A or B Arm in the Heterodimeric Vaccine Protein (A) BALB/c mice were vaccinated with 100 μg (two plasmids) of DNA i.m./EP with A/B heterodimers expressing influenza HA antigen from Cal07 (H1N1) and PR8 (H1N1) connected to either the A or B arm (n = 5-6/group). 14 days after vaccination, the mice were bled and Cal07- and PR8-specific IgGs were analyzed in the sera by ELISA (mean ± SEM; Mann-Whitney test, ns, not significant; ND, not detectable). (B) BALB/c mice were vaccinated with 50 μg (two plasmids) of DNA i.d./EP with A/B heterodimers expressing influenza HA antigen from PR8 (H1N1) and OVA connected to either the A or the B arm (n = 4/group, analyzed as biological replicates). After 11 days, spleens were harvested and analyzed for IFN-γ-secreting T cells (ELISPOT) after stimulation with HA whole protein, MHCII-restricted HA peptides (HNTNGVTAACSHEG or SVSSFERFEIFPK), or MHCI-restricted HA peptide (IYSTVASSL), OVA whole protein, MHCII-restricted OVA peptide (ISQAVHAAHAEINEAGR), or irrelevant peptide (λ2 315 CDR3 peptide, ALWFRNHFVFGGGTKVT). Mann-Whitney test, two-tailed; ns, not significant.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Enzyme-linked Immunospot, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza

doi: 10.1016/j.omtm.2020.01.007

Figure Lengend Snippet: Comparison of A/B Heterodimeric and C H 3-Homodimeric DNA Vaccines Expressing Two Different HAs BALB/c mice were vaccinated i.m./EP with 100 μg of DNA (two plasmids) encoding either A/B heterodimeric or C H 3 homodimeric vaccine proteins that express HA from both PR8(H1N1) and Cal07(H1N1) as indicated. Mice were boosted after 5 weeks. (A and B) Levels of PR8-specific IgG (A) and Cal07-specific IgG (B) serum antibodies 2 weeks after the boost (mean ± SEM; **p < 0.01 Mann Whitney; ND, not detectable). (C and E) 2 weeks after the boost, the mice were infected with a lethal dose of PR8 (C, 5 × LD 50 ), or Cal07 (E, 5 × LD 50 ) influenza virus. Weight was followed for 10 days. Weight loss of 20% was defined as the humane endpoint, at which time point the mice were euthanized (n = 8/group, mean ± SEM; Mann-Whitney test, *p < 0.05, **p < 0.01; ns, not significant). (D and F) Survival curves of the vaccinated mice after challenge with either PR8 (D) or Cal07 (F) influenza virus.

Article Snippet: Spleens were harvested after 14 days, plated in triplicates (5 × 10 5 cells/well), and stimulated with 2 μg/mL MHCII (SVSSFERFEIFPK and HNTNGVTAACSHEG)- or MHCI (IYSTVASSL, both InvivoGen)-restricted HA PR8 peptides, MHCII-restricted OVA peptide (amino acids 323–339, ISQAVHAAHAEINEAGR, InvivoGen), or complete recombinant HA PR8 protein (2 μg/mL, Sino Biological) or OVA protein (5 μg/mL, InvivoGen).

Techniques: Expressing, MANN-WHITNEY, Infection

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and bronchial epithelial cell viability. Medium release of lactate dehydrogenase (LDH) was measured and expressed as % viability in BEAS-2B cells ( A ) or LDH activity in 16HBE cells ( B ) after 24 h treatment with 0.1 µM to 10 mM ADX-102 in M-199 with 10% serum (Media). An equal number of cells were sonicated for maximum LDH release (Lysed). Positive assay control (+) was provided by a manufacturer. * p < 0.05 and ** p < 0.01 vs. 10 µM ADX-102; **** p < 0.0001 vs. 0–100 µM ADX-102. Bars represent SEM of biological n of three with three technical replicates. ns = not significant.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques: Activity Assay, Sonication

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and bronchial epithelial cell migration. Circular wounds in monolayers of BEAS-2B cells were measured for cell migration into the wound area in the presence of 0–100 µM ADX-102 over time ( A ). Positive control for wound closure was M-199 with 10% serum (FBS). In panel ( B ), BEAS-2B cells were wounded in the presence of 5% cigarette smoke extract (CSE) and in the presence of 0–100 µM ADX-102, and % wound closure was measured. * p < 0.05 vs. 0 µM ADX-102. Bars represent SEM of n = 5, each with three replicates.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques: Migration, Positive Control

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and bronchial epithelial cell protein kinase C alpha (PKCα) activity. BEAS-2B cells were pretreated with or without 10 µM ADX-102 for 1 h prior to treatment with M-199 containing 10% serum (Media) or 5% cigarette smoke extract (CSE) for 1 h. In the absence of ADX-102, **** p < 0.0001 CSE vs. Media. In the presence of CSE, ** p < 0.01. No ADX vs. ADX. Bars represent SEM of n = 9, each with three replicates.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques: Activity Assay

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and tracheal epithelial ciliated cell protein kinase C epsilon mediated cilia beating. Ciliated MTECs were treated with or without the combination of 5% cigarette smoke extract (CSE) and 50 mM ethanol (EtOH) in the presence or absence of 10 µM ADX-102, and cilia beat frequency (CBF) was measured from 0–24 h ( A ). Baseline control consisted of DMEM with 10% serum (Media). * p < 0.05 ADX vs. no ADX in the presence of CSE + EtOH at 3, 6, and 24 h. Dose–response (0–100 µM) for ADX-102 on combined CSE and EtOH stimulated (3 h) and autodownregulated (24 h) protein kinase C epsilon (PKCε). * p < 0.01 vs. medium control. Bars represent SEM of n = 6, each with three replicates ( B ).

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques:

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and loss of tracheal epithelial cell cilia. Ciliated MTECs were treated with a combination of 5% cigarette smoke extract (CSE) and 50 mM ethanol (EtOH) in the presence or absence of 10 µM ADX-102, and the average number of motile cilia were measured at 3 ( A ) and 24 h ( B ). **** p < 0.01 vs. medium control or presence of ADX. Bars represent SEM of at least n = 9 individual experiments. ns = not significant.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques:

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and bronchial epithelial cell permeability. 16HBE cells were grown to confluence until maximal barrier function (Resistance) achieved. Cells were treated with either DMEM and 10% serum (Media), 5% cigarette smoke extract (CSE), 50 mM alcohol (EtOH), or the combination of smoke and alcohol for up to 72 h in the absence ( A ) or presence ( B ) of 10 µM ADX-102. * p < 0.02 vs. media at 72 h. Bars represent SEM of n = 5, each with three replicates.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques: Permeability

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: ADX-102 and MAA adduct formation. 16HBE cells were grown to confluence in 60 mm culture dishes. Cells were treated with either M-199+10% serum (Media) or a combination of 5% cigarette smoke extract (CSE) and 50 mM alcohol (EtOH) for 72 h in the absence or presence of 10 µM ADX-102. * p <0.04 and ** p < 0.003 at 72 h. Bars represent SEM of n = 6.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques:

Journal: Biomolecules

Article Title: Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

doi: 10.3390/biom12030393

Figure Lengend Snippet: Model diagram for ADX-102 aldehyde-trapping action on cigarette smoke and alcohol mediated injury to airway epithelial cell function.

Article Snippet: They were grown to confluency and treated with different doses of cigarette smoke or ethanol in the presence or absence of ADX-102 (TargetMol, Wellesley Hills, MA, USA) for a maximum of 72 h. Controls consisted of medium-only wells with and without cells.

Techniques: Cell Function Assay

Journal: Molecular Genetics & Genomic Medicine

Article Title: Further support linking the 22q11.2 microduplication to an increased risk of bladder exstrophy and highlighting LZTR1 as a candidate gene

doi: 10.1002/mgg3.666

Figure Lengend Snippet: Lztr1 wt and Lztr1 mut differ in their intracellular distribution in live NIH 3T3 cells. CLSM images of the spatial distribution of Lztr1 wt (a and d), and Lztr1 mut (b and e), genetically fused with the reporter molecule TurboGFP, in live NIH 3T3 cells CLSM reveals that both Lztr1 wt and Lztr1 mut are localized in distinct, spatially confined structures that are associated with the endomembrane system (consistent with the Golgi), but Lztr1 wt was also observed in the cytoplasm, whereas Lztr1 mut was not. Images (c and f) show the uniform intracellular distribution of the fluorescence reporter, TurboGFP, alone

Article Snippet: LZTR1 (NM_006767.3) human cDNA ORF construct tagged with C‐termial GFP‐tag in pCMV6‐AC‐GFP vector was purchased (RG204432, Origene, MD, USA).

Techniques: Fluorescence

Journal: Molecular Genetics & Genomic Medicine

Article Title: Further support linking the 22q11.2 microduplication to an increased risk of bladder exstrophy and highlighting LZTR1 as a candidate gene

doi: 10.1002/mgg3.666

Figure Lengend Snippet: Lztr1 wt and Lztr1 mut differ in their intracellular distribution and Lztr1 wt mobility decay shifts to longer lag times with concentration, indicating complex formation in live NIH 3T3 cells. (a) Fluorescence intensity fluctuations recorded in the cytoplasm of live cells expressing Lztr1 wt (blue) or Lztr1 mut (wine). Fluorescence intensity fluctuations recorded in the cell culture medium (cyan) are shown for comparison. (b) Temporal autocorrelation curves (tACC) recorded in the cytoplasm of live cells expressing Lztr1 wt at different levels (10–70 nmol L ‐1 ). Temporal autocorrelation analysis of fluorescence intensity fluctuations recorded in the cytoplasm of cells expressing Lztr1 mut showed that Lztr1 mut is not distributed in the cytoplasm (wine), outside of the very bright speckles observed by imaging. Nuclear localization was not observed; neither for Lztr1 wt nor for Lztr1 mut (dark green). (c) tACCs normalized to the same amplitude, G n (τ) = 1 at = τ10 µs, show that Lztr1 wt mobility is much slower compared to the mobility of TurboGFP (dark yellow), as evident from the shift of the characteristic decay time of the tACCs for Lztr1 wt towards longer lag times. FCS analysis also revealed that Lztr1 wt self‐assembles into larger supra‐molecular complexes when expressed at higher levels, as evident from the shift of the characteristic decay time of the tACC toward longer lag times. In addition, the contribution of the slower component increases for increasing Lztr1 wt concentration

Article Snippet: LZTR1 (NM_006767.3) human cDNA ORF construct tagged with C‐termial GFP‐tag in pCMV6‐AC‐GFP vector was purchased (RG204432, Origene, MD, USA).

Techniques: Concentration Assay, Fluorescence, Expressing, Cell Culture, Imaging

Journal: Frontiers in Immunology

Article Title: Baicalin Induces a Potent Innate Immune Response to Inhibit Respiratory Syncytial Virus Replication via Regulating Viral Non-Structural 1 and Matrix RNA

doi: 10.3389/fimmu.2022.907047

Figure Lengend Snippet: Oligonucleotides used for RT-qPCR.

Article Snippet: Anti-NS1, anti-NS2, and anti-M antibodies were produced by AtaGenix (Wuhan, China).

Techniques:

Journal: Frontiers in Immunology

Article Title: Baicalin Induces a Potent Innate Immune Response to Inhibit Respiratory Syncytial Virus Replication via Regulating Viral Non-Structural 1 and Matrix RNA

doi: 10.3389/fimmu.2022.907047

Figure Lengend Snippet: Baicalin demonstrates potent anti-RSV activity in vitro . (A) The cytotoxic influence of baicalin was determined by assessing cell viability by CCK-8 assay. HEp-2 cells were treated in triplicate with baicalin increased concentrations for 72 h. Absorbance at 570 nm in relation to that of mock treated control was plotted for evaluation of the TC 50 value. (B) Expression of viral transcripts of the NS2 gene was analyzed by RT-qPCR in HEp-2 cells infected with RSV (MOI = 3) and treated with the indicated doses of baicalin. Data were normalized to Actb expression. Results are shown as mean ± SD (n = 3); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 – ANOVA with Tukey’s post-hoc test. (C, D) Baicalin IC 50 was determined through plaque reduction assessment in HEp-2 cells infected with RSV in the presence of the indicated doses of baicalin (C) . The number of plaques was counted and plotted as the percentage of plaque inhibition in relation to untreated cells, after 72 h (D) .

Article Snippet: Anti-NS1, anti-NS2, and anti-M antibodies were produced by AtaGenix (Wuhan, China).

Techniques: Activity Assay, In Vitro, CCK-8 Assay, Control, Expressing, Quantitative RT-PCR, Infection, Inhibition

Journal: Frontiers in Immunology

Article Title: Baicalin Induces a Potent Innate Immune Response to Inhibit Respiratory Syncytial Virus Replication via Regulating Viral Non-Structural 1 and Matrix RNA

doi: 10.3389/fimmu.2022.907047

Figure Lengend Snippet: Baicalin affects transcriptional and translational regulation of RSV gene products. (A) Baicalin causes the downregulation of viral genes NS1 and NS2, but not others. Expression of indicated viral transcripts in lung homogenates of RSV-infected mice ± baicalin was quantified by RT-qPCR. Outcomes were normalized to Actb . (B) Immunoblot analysis of NS1, NS2, and M protein in lung homogenates of RSV-infected mice ± baicalin showed downregulation in the expression of these proteins after treatment with baicalin. Representative blots from 3 animals are shown. β-actin was immunobloted as a loading control. (C) Polysome occupancy of NS1, NS2, and M RNAs. Baicalin caused translational downregulation of viral M RNA. The decrease in polysome occupancy observed for NS1 and NS2 is attributable to the decrease in their total RNA. Data shown in (A , C) represents mean ± SD (n = 5); *** P < 0.001, **** P < 0.0001, ns , not significant – ANOVA with Tukey’s post-hoc test.

Article Snippet: Anti-NS1, anti-NS2, and anti-M antibodies were produced by AtaGenix (Wuhan, China).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Baicalin Induces a Potent Innate Immune Response to Inhibit Respiratory Syncytial Virus Replication via Regulating Viral Non-Structural 1 and Matrix RNA

doi: 10.3389/fimmu.2022.907047

Figure Lengend Snippet: Model summarizing mechanism underlying potent innate immune response elicited against RSV infection by treatment with baicalin. Translational regulation of RSV viral M RNA was previously elucidated . Briefly, baicalin suppresses translation of the viral M protein and transcription of the viral NS1 and NS2 proteins and downregulates the expression of pro-inflammatory cytokines while inducing expression of type I IFNs, thus mounting a potent anti-RSV innate immune response.

Article Snippet: Anti-NS1, anti-NS2, and anti-M antibodies were produced by AtaGenix (Wuhan, China).

Techniques: Infection, Expressing

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

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

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

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

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

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

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