α human anp32a  (Proteintech)

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: Human ANP32A and ANP32B are required host factors for thogotovirus polymerase activity and replication. ( A–F ) ANP32A and ANP32B increase polymerase activity for diverse orthomyxoviruses. Polymerase activity assays were performed in WT or knockout cells by expressing the polymerase proteins, nucleoprotein, and a viral firefly luciferase reporter for the indicated virus. Polymerase activity was normalized to an internal Renilla luciferase control and reported as the ratio of firefly/Renilla luciferase. FLUCV, influenza C virus. The polymerase subunit PB2 was expressed with a FLAG epitope tag. PB2 and the loading control β-actin were detected by western blotting. ( G and H ) Multicycle replication of BRBV (multiplicity of infection [MOI] = 0.1) or DHOV (MOI = 1) in 293T and knockout cells. Titers determined by focus-forming assay. LOD, limit of detection. ( A–F ) Data are mean of n = 3 ± sd and significance was assessed by a one-way analysis of variance (ANOVA) with a post hoc Dunnett’s multiple comparisons test against WT 293T cells. ( G and H ) Data are mean of n = 6 ± sd with comparisons to DKO cells made by a two-way ANOVA with a Tukey’s post hoc test. ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Knock-Out, Expressing, Luciferase, Virus, Control, FLAG-tag, Western Blot, Infection, Focus Forming Assay

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: Human ANP32A or ANP32B is sufficient for thogotovirus polymerase activity and replication. ( A–F ) Polymerase activity was measured in WT 293T cells, DKO cells, or DKO cells stably complemented with human ANP32A or ANP32B. Polymerase activity assays were performed by expressing the polymerase proteins, nucleoprotein, and a viral firefly luciferase reporter for the indicated virus. Polymerase activity was normalized to an internal Renilla luciferase control. ( G and H ) Human ANP32A and ANP32B enhance BRBV polymerase activity in a dose-dependent manner. Polymerase activity was measured in DKO cells as in ( A ) in the presence of increasing amounts of expression vector for ( G ) human ANP32A or ( H ) human ANP32B. PB2-FLAG, ANP32-FLAG, and the loading control β-actin were detected by western blotting. ( I ) Multicycle replication of BRBV in 293T cells, DKO cells, or DKO cells stably complemented with human ANP32A or human ANP32B (MOI = 0.01). Titers were determined by focus-forming assay. ( A–H ) Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against WT 293T cells ( A–F ) or empty vector controls ( G and H ). ( I ) Data are mean of n = 6 ± sd with comparisons to DKO cells made by a two-way ANOVA with a Tukey’s post hoc test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Stable Transfection, Expressing, Luciferase, Virus, Control, Plasmid Preparation, Western Blot, Focus Forming Assay

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: BRBV polymerase binds ANP32 and requires its C-terminal low complexity acidic region. ( A ) Schematic diagram of full-length human ANP32B and three C-terminal truncations at amino acid 160 (T160), 190 (T190), and 220 (T220). Domains and boundaries are indicated. LRR = leucinerich repeat; LCAR = low complexity acidic region. T160, T190, and T220 = terminal amino acids in C-terminal truncations. ( B ) Human ANP32A (hu32A) interacts with the BRBV polymerase trimer. Human ANP32A-FLAG was expressed in cells with the BRBV polymerase or versions lacking PB2 or PA. Human ANP32A-FLAG was immunoprecipitated from whole-cell lysate and probed for huANP32A or V5-tagged PB2 and PA. Input samples were blotted as an expression control. β-actin served as a loading control. ( C ) The LCAR of human ANP32B (hu32B) is important for interaction with the BRBV polymerase. Polymerase proteins were expressed in DKO cells in the presence of human ANP32B-FLAG, the indicated truncations, or a negative control. Interaction with the polymerase was tested by immunoprecipitating huANP32B-FLAG from whole-cell lysate and probing for huANP32B and co-precipitating PB2 and PA that contained a C-terminal V5 tag. Input samples were blotted as an expression control. β-actin served as a loading control. ( D ) BRBV polymerase activity assays were performed in DKO cells expressing WT or truncated huANP32B, or an empty vector control. Polymerase activity was normalized to an internal Renilla luciferase control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against cells expressing WT huANP32B. **** P ≤ 0.0001.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Immunoprecipitation, Expressing, Control, Negative Control, Activity Assay, Plasmid Preparation, Luciferase

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: Ticks encode a single ancestral locus expressing ANP32A variants that support BRBV polymerase activity. ( A ) Syntenic gene blocks were identified for ANP32 genes, revealing a single ancestral locus present in the lone star (Aam) ticks and brown dog (Rsa) ticks. Comparisons were made to humans (Hsa, Homo sapiens ), chickens (Gga), swine (Ssc), and cows (Bta). ( B ) De novo transcriptome assembly identifies multiple splice variants of tick ANP32. The genomic locus for Rsa and Aam ticks is indicated, as well as accession numbers for previously cataloged transcript variants. Exons are shown as boxes with non-coding regions in pink, open reading frames in red, and a small upstream open reading frame (uORF) in gray. The conserved start site present in vertebrate ANP32A is indicated. ( C ) BRBV polymerase activity assays were performed in DKO cells expressing the viral polymerase, nucleoprotein, and a viral reporter as well as the indicated ANP32A or ANP32B from humans (hu), cows (co), chickens (ch), lone star ticks (Aam), or brown dog ticks (Rsa). Naming of tick variants corresponds to those diagrammed in (B). Activity was normalized to an internal Renilla luciferase control. ( D ) Polymerase activity assays were performed for DHOV and THOV as in (C). ( E ) Activity of the FLUAV polymerase in cells expressing diverse ANP32 proteins was measured as described for (C). For (C)–(E), data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against the empty vector. ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant. ( F ) Expression of ANP32 proteins was detected by western blot. Tubulin was probed as a loading control.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Expressing, Activity Assay, Luciferase, Control, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: BRBV polymerase activity is sensitive to changes at the N-terminus of ANP32. ( A ) Alignment of the N-terminus of human ANP32A (hu32A), ANP32B (hu32B), and lone star tick (Aam) ANP32A (Aam 32). Mutants used below are highlighted in red. ( B–D ) Changes to the N-terminus of ANP32 disrupt its ability to support BRBV polymerase. ( B–G ) Polymerase activity assays for the indicated polymerase were performed in DKO cells expressing huANP32A with a C-terminal FLAG tag (C-FLAG), an N-terminal FLAG tag (N-FLAG), a C-terminal FLAG tag with an N-terminal deletion (∆ME) or mutation (E2A), or an empty vector control. Polymerase lacking the PB2 subunit (∆PB2) served as a negative control. ( H–I ) Polymerase activity assays were performed with the indicated polymerase in DKO cells expressing huANP32B with a C-terminal FLAG tag (C-FLAG), an N-terminal FLAG tag (N-FLAG), a C-terminal FLAG tag with an N-terminal deletion (∆MD) or mutation (D2A), or an empty vector control. Polymerase lacking the PB2 subunit (∆PB2) served as a negative control. For all, polymerase activity was normalized to an internal Renilla luciferase control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against huANP32 C-FLAG. **** P ≤ 0.0001; ns, not significant. ( J ) Representative blot of ANP32 proteins and variants confirming equivalent expression. Tubulin was probed as a loading control.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Expressing, FLAG-tag, Mutagenesis, Plasmid Preparation, Control, Negative Control, Luciferase

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: The impact of adaptive variants in chicken ANP32A and ANP32B differs between BRBV and FLUAV polymerases. ( A–D ) Polymerase activity assays were performed in DKO expressing the indicated viral polymerase, nucleoprotein, and a viral reporter. Cells were complemented with human (huANP32), chicken (chANP32), chANP32A lacking residues 175-207 (ch∆33), or mutant huANP32B N129I/D130N proteins. Activity was normalized to an internal Renilla luciferase control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons. ANP32A variants were compared against huANP32A, and ANP32B variants were compared against huANP32B. * P ≤ 0.05; *** P ≤ 0.001; ns, not significant. ( E ) Representative blot confirming expression of ANP32 variants. Tubulin was detected as a loading control.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Expressing, Mutagenesis, Luciferase, Control

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: Amino acid variations in the N-terminus and central regions of ANP32E preclude its use by BRBV or FLUAV, respectively. ( A ) Diagram of chimeric proteins between human ANP32A and ANP32E. Fusions were made after residues 120 and 170. ( B ) BRBV polymerase, nucleoprotein, and a viral reporter were expressed in DKO cells with the indicated human ANP32A, ANP32E, or chimeric clone. Activity was normalized to an internal Renilla luciferase control. ( C ) FLUAV polymerase activity was measured as in (B). ( D ) Representative blot showing expression of ANP32 proteins and chimeras. Tubulin was detected as a loading control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against human ANP32A or ANP32E. *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Luciferase, Control, Expressing

Journal: Biomedicines

Article Title: The Effect of Tff3 Deficiency on the Liver of Mice Exposed to a High-Fat Diet

doi: 10.3390/biomedicines13051024

Figure Lengend Snippet: Visualization of protein interactions that were statistically significantly altered in livers of Tff3 −/− females compared to WT females fed on high-fat diet. Interactions were visualised using programme tool STRING . Relative expression changes are represented by blue circle around protein (gradation from upregulated (dark blue) to downregulated level (pale blue circle). Abbreviation: Aldh1a7—aldehyde dehydrogenase, cytosolic 1; Anp32a—acidic nuclear phosphoprotein pp32; Dpp4—dipeptidyl peptidase 4 isoform 1; Ech1—enoyl coenzyme A hydratase 1, peroxisomal; Fah—fumarylacetoacetase hydrolase; Gchfr—GTP cyclohydrolase I feedback regulator; H2afv—histone H2afv; H2afx—histone H2A.X; Hacl1—2-hydroxyacyl-CoA lyase 1; Hist1h2bp—histone H2B type 1-P isoform 1; Hmgcs1—3-hydroxy-3-methylglutaryl-coenzyme A synthase 1; Hnrnpd—heterogeneous nuclear ribonucleoprotein AU-rich element RNA-binding protein 1; Mbl2—mannose-binding protein C precursor; Nolc1—nucleolar and coiled-body phosphoprotein 1; Pdia4—protein disulfide-isomerase A4 isoform 1 precursor; Picalm—phosphatidylinositol binding clathrin assembly protein; Ppib—peptidylprolyl isomerase B; Rpl17—ribosomal protein; Rpl19—ribosomal protein L19; Rpl26—60S ribosomal protein L26; Rpl28—60S ribosomal protein L28 isoform X1; Sec14l2—SEC14-like protein 2; Sfpq—splicing factor proline/glutamine-rich polypyrimidine tract binding-associated protein; Uqcrq—ubiquinol-cytochrome c reductase, complex III subunit VII; Vapb—vesicle-associated membrane protein, associated protein B and C; Ybx1—Y-box binding protein.

Article Snippet: Then, a 5% I-BlockTM protein-based blocking reagent (T2015) in 1× PBS was used to block non-specific binding for 1 h. Membranes were incubated overnight at 4 °C with anti-Ybx1 (ab76149, Abcam, Cambridge, UK) and anti-Anp32a primary antibodies (15810-1-AP, Proteintech, Manchester, UK), diluted 1:1000.

Techniques: Expressing, RNA Binding Assay, Binding Assay, Membrane

Journal: Biomedicines

Article Title: The Effect of Tff3 Deficiency on the Liver of Mice Exposed to a High-Fat Diet

doi: 10.3390/biomedicines13051024

Figure Lengend Snippet: Ybx1 and Anp32a protein levels were significantly upregulated in the liver of Tff3 −/− females compared to WT female. Proteins were isolated from the liver tissue of mice exposed to a high-fat diet and analysed by Western blotting. ( a ) The protein level of Ybx1 and Anp32a in the liver of WT and Tff3 −/− females exposed to high-fat diet (HFD) analysed by the Western blot method using specific antibodies. ( b ) Results were quantified using the Image J programme, and signals were normalised using amidoblack staining. Student’s t test was used for statistical analysis of the obtained values and the results are presented as the mean value and standard deviation. Statistical significance is shown: * p ≤ 0.05, ** p ≤ 0.01. Symbols: ♀—female.

Article Snippet: Then, a 5% I-BlockTM protein-based blocking reagent (T2015) in 1× PBS was used to block non-specific binding for 1 h. Membranes were incubated overnight at 4 °C with anti-Ybx1 (ab76149, Abcam, Cambridge, UK) and anti-Anp32a primary antibodies (15810-1-AP, Proteintech, Manchester, UK), diluted 1:1000.

Techniques: Isolation, Western Blot, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Polymerase mutations underlie early adaptation of H5N1 influenza virus to dairy cattle and other mammals

doi: 10.1101/2025.01.06.631435

Figure Lengend Snippet: A) Key mammalian adaptations identified in this study mapped to the structure of H5N1 influenza A replicase dimer complex with human ANP32B (PDB: 8R1J). ANP32B is shown in cyan, PB1 in purple, PA in violet and PB2 as a salmon ribbon representation. H5N1 cattle PB2 substitutions (Q591, E627, M631 and D740) and PA (K497R) shown in red. B-F) Minigenomes in human engineered haploid cells (eHAP) with endogenous ANP32A, ANP32B and ANP32E knocked out were supplemented with ANP32 proteins from different species (chANP32A = chicken ANP32A, swANP32A = swine ANP32A, swANP32B = swine ANP32B, bovANP32A = bovine ANP32A, bovANP32B = bovine ANP32B, huANP32A = human ANP32A, huANP32B = human ANP32B, -ANP = no ANP32 control). Avianised cattle = PB2 M631L and PA K497R reversed. Data normalised throughout to chANP32A. Data throughout plotted as triplicate technical repeats of a representative repeat of N = 3 independent repeats. Data throughout plotted as mean + SD. Statistics throughout performed by one-way ANOVA with multiple comparisons - against -ANP32 control, or between species-matched ANP32A and ANP32B proteins, using log-transformed data. Log-normality determined by Shapiro-Wilk test and QQ plot. Significance shown by asterisks indicating: *, 0.05□≥□P□>□0.01; **, 0.01□≥□P□>□0.001; ***, 0.001□≥□P□>□0.0001; ****, P□≤□0.0001.

Article Snippet: Bos taurus Anp32A (NCBI reference sequence NM_001195019.1) and Anp32B (NM_001035074.1) were ordered as synthetic GeneArt constructs from ThermoFisher Scientific and subcloned into pCAGGS as described previously .

Techniques: Control, Transformation Assay