Journal: Heliyon

Article Title: Host proteins interact with viral elements and affect the life cycle of highly pathogenic avian influenza A virus H7N9

doi: 10.1016/j.heliyon.2024.e28218

Figure Lengend Snippet: Co-immunoprecipitation (Co-IP) and immunoblotting analysis of the interactions between targeted host proteins and HP001 proteins. Extracts from A549 cells infected with HP001 were incubated with antibodies recognizing targeted host proteins plus Protein G beads; proteins pulled down were detected using western blotting using anti-HA, M1, NA, NP, PA, and PB1 antibodies. Extracts containing HP001 incubated with Protein G beads without any antibody, and extracts from normal cells without HP001 mixed with Protein G beads and antibodies served as controls. Isotype antibodies served as negative controls. (A) Six host proteins (ANXA2, ANXA5, ATP5A1, COPA, HSPA1A, and HSPA8) interacted with HA. (B) Seven host proteins (AP2S1, AP3S1, COPA, COPG1, HSPA1A, HSPA8 and RAB18) interacted with M1. (C) Six host proteins (ANXA2, ANXA5, AP2S1, AP3S1, COPA and HSP90AA1) interacted with NA. (D) Seven host proteins (ANXA2, AP2S1, AP3S1, COPA, HSPA1A, HSPA8, and RAB18) interacted with NP. (E) Four host proteins (ANXA2, HSPA1A, RAB11B and RAB18) interacted with PA. (F) Four host proteins (ATP5A1, HSPA1A, HSP90AA1 and RAB11B) interacted with PB1. HA, hemagglutinin; M1, matrix 1 protein; NA, neuraminidase; NP; nucleoprotein; PA, polymerase basic 1; PB1, polymerase basic 2; ANXA2, annexin A2; ANXA5 annexin A5; ATP5A1, ATP synthase F1 subunit alpha; COPA, COPI coat complex subunit alpha; HSPA1A, heat shock protein family A (Hsp70) member 1A; HSPA8, heat shock protein family A (Hsp70) member 8; AP2S1, AP3S1, COPG1, COPI coat complex subunit G1; HSP90AA1, heat shock protein 90 alpha family class A member 1.

Article Snippet: Antibodies recognizing Annexin A (ANXA)2 (Cat# NB100-2724), heat shock protein family A (Hsp70) member 1A (HSPA)1A (Cat# NB120-2788), HSPA8 (Cat# NBP2-12880), influenza A virus PA (Cat# NBP2-42876), and influenza A virus PB1 (Cat# NBP2-42877) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Infection, Incubation

Journal: Virologica Sinica

Article Title: Equine ANP32 proteins support influenza A virus RNA polymerase activity

doi: 10.1016/j.virs.2023.10.009

Figure Lengend Snippet: Support of equine influenza viral replication by equine ANP32A or ANP32B, and species-dependent support of equine ANP32A or B for influenza A viral replication. Vectors carrying 20 ​ng of ANP32A or ANP32B genes, or empty vectors, were co-transfected into DKO cells (a 293T knockout cell line), together with a minigenome reporter, a Renilla expression control, and influenza virus polymerases from either equine influenza H3N8 XJ07 ( A ); human influenza H7N9 AH13 ( B ); canine influenza H3N2 GD11 ( C ); swine influenza H1N1 NC08 ( D ); avian influenza H9N2 ZJ12 ( E ). Luciferase activity was measured 20 ​h later, and data indicate the firefly luciferase gene activity normalized to the Renilla luciferase gene activity. Statistical differences between samples are indicated, according to a one-way ANOVA, followed by a Dunnett's test (ns, not significant; ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001). Error bars represent the SEM within one representative experiment. The results represent at least three independent experiments. Western blotting to detect the ANP32A and viral proteins used specific antibodies: PA antibody and PB2 antibody (from our lab), PB1 antibody (NBP2-42877, NOVUS), Anti-Flag antibody (F1804, SIGMA) and Anti-β-actin antibody (F1804, SIGMA). ch, chicken; sw, swine; hu, human; eq, equine.

Article Snippet: Western blotting to detect the ANP32A and viral proteins used specific antibodies: PA antibody and PB2 antibody (from our lab), PB1 antibody (NBP2-42877, NOVUS), Anti-Flag antibody (F1804, SIGMA) and Anti-β-actin antibody (F1804, SIGMA). ch, chicken; sw, swine; hu, human; eq, equine.

Techniques: Transfection, Knock-Out, Expressing, Control, Virus, Luciferase, Activity Assay, Western Blot

Journal: Virologica Sinica

Article Title: Equine ANP32 proteins support influenza A virus RNA polymerase activity

doi: 10.1016/j.virs.2023.10.009

Figure Lengend Snippet: The N-Cap domain does not affect the interaction of eqANP32A with the trimeric equine influenza virus polymerase complexes. A DKO cells (a 293T knockout cell line) were transfected with different ANP32A (0.6 ​μg) and polymerase plasmids (0.6 ​μg ​PA, 1 ​μg PB1, and 1 ​μg PB2) from equine influenza virus H3N8 XJ07 . The cells were lysed at 36 ​h post-transfection. Co-IP was performed using anti-FLAG M2 magnetic beads, followed by Western blotting to detect the ANP32A and viral proteins using specific antibodies: PA antibody and PB2 antibody (from our lab), PB1 antibody (NBP2-42877, NOVUS), Anti-Flag antibody (F1804, SIGMA) and Anti-β-actin antibody (F1804, SIGMA). To compare the binding ability of eqANP32 with each of the polymerase protein, the intensity of each protein band was measured using the ImageJ software. The intensity bands ratio was the value of the intensity of each polymerase protein divided by that of eqANP32 ( B and C ). P values were determined using one-way ANOVA followed by a Dunnett's multiple comparisons test. ns, not significant; ∗, P ​< ​0.05; ∗∗, P ​< ​0.01. eq, equine.

Article Snippet: Western blotting to detect the ANP32A and viral proteins used specific antibodies: PA antibody and PB2 antibody (from our lab), PB1 antibody (NBP2-42877, NOVUS), Anti-Flag antibody (F1804, SIGMA) and Anti-β-actin antibody (F1804, SIGMA). ch, chicken; sw, swine; hu, human; eq, equine.

Techniques: Virus, Knock-Out, Transfection, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Binding Assay, Software

Journal: Virologica Sinica

Article Title: Equine ANP32 proteins support influenza A virus RNA polymerase activity

doi: 10.1016/j.virs.2023.10.009

Figure Lengend Snippet: Differences in ANP32B support the activity of viral polymerases from influenza viruses infecting different species. DKO cells were co-transfected with expression plasmids carrying PB1 (40 ​ng), PB2 (40 ​ng), PA (20 ​ng) and NP (80 ​ng) from equine influenza H3N8 XJ07 ( A ); canine influenza H3N2 GD11 ( B ), together with 40 ​ng minigenome reporter and 1 ​ng Renilla luciferase expression plasmids (pRL-TK, as an internal control) in the presence of chANP32B, swANP32B, eqANP32B, huANP32B or caANP32B protein or empty vector. Cells were then lysed using passive lysis buffer, and luciferase activity was measured at 20 ​h post transfection. Statistical differences between samples are indicated, according to a one-way ANOVA, followed by a Dunnett's test (ns, not significant; ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001). Error bars represent the SEM within one representative experiment. The results represent at least three independent experiments. ch, chicken; sw, swine; hu, human; ca, canine.

Article Snippet: Western blotting to detect the ANP32A and viral proteins used specific antibodies: PA antibody and PB2 antibody (from our lab), PB1 antibody (NBP2-42877, NOVUS), Anti-Flag antibody (F1804, SIGMA) and Anti-β-actin antibody (F1804, SIGMA). ch, chicken; sw, swine; hu, human; eq, equine.

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Control, Plasmid Preparation, Lysis

Journal: Journal of reproductive immunology

Article Title: Complement activation by an angiogenic imbalance leads to systemic vascular endothelial dysfunction: A new proposal for the pathophysiology of preeclampsia.

doi: 10.1016/j.jri.2021.103322

Figure Lengend Snippet: Fig. 1. CFH expression and viability of HUVECs transfected with CFH-siRNA. (a) CFH expression was lower in HUVECs transfected with specific siRNA (100 nM) than in controls. Quantitative RT-PCR showed that CFH expression was knocked down by 80 % using specific siRNA (n = 5, P < 0.05). (b) As determined by a cell viability assay, HUVECs transfected with CFH-siRNA were more vulnerable than control cells after incubation with com plement medium, as seen by the lower viability (n = 15; P < 0.05).

Article Snippet: HUVECs were plated at a density of 1 × 104 cells/ well in 96-well plates in triplicate and cultured for 24 h. The cells were then transfected for 6 h with 100 nM CFH-specific siRNA (sc-42,877) or control siRNA-A (sc-37,007) using a transfection reagent (sc-29,528), according to the manufacturer’s instructions (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Viability Assay, Control, Incubation

Journal: PLoS Pathogens

Article Title: A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs

doi: 10.1371/journal.ppat.1008330

Figure Lengend Snippet: DKO cells were transfected with different ANP32A (0.6μg) and polymerase plasmids (0.6μg PA, 1μg PB1, and 1μg PB2) from avian influenza viruses H7N9 ZJ13 (A) , H9N2 ZJ12 (B) , human influenza virus polymerase WSN (C) . The cells were lysed at 24 h post-transfection. Co-IP was performed using Anti-FLAG M2 Magnetic Beads, followed by Western blotting to detect the ANP32A and viral proteins by using specific antibodies: PA antibody (NBP2-42874, NOVUS), PB1 antibody (NBP2-42877, NOVUS), PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).

Article Snippet: This experiment was carried out using the following primary antibodies: Influenza A Virus PA antibody (NBP2-42874, NOVUS), Influenza A Virus PB1 antibody (NBP2-42877, NOVUS), Influenza A Virus PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).

Techniques: Transfection, Virus, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: PLoS Pathogens

Article Title: A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs

doi: 10.1371/journal.ppat.1008330

Figure Lengend Snippet: (A) Schematic of alignment from mammalian ANP32A amino acid sequences including human ANP32A (huANP32A), canine ANP32A (caANP32A), equine ANP32A (eqANP32A) and swine ANP32A (swANP32A). Three residues were annotated as there are mutations on swANP32A, at positions 106, 156, and 228. (B, C) The mutants of swANP32A were constructed by overlapping PCR. DKO cells were co-transfected with expression plasmids carrying PB1 (40 ng), PB2 (40 ng), PA (20 ng) and NP (80 ng) from H7N9 ZJ13 , together with 40 ng minigenome reporter and 10 ng Renilla luciferase expression plasmids (pRL-TK, as an internal control) in the presence of swANP32A or its mutants or empty vector. Cells were then lysed using passive lysis buffer and luciferase activity was measured at 24 h post transfection. (D) The mutants of huANP32A were co-transfected into DKO cells together with polymerase plasmids from H7N9 ZJ13 , plus a minigenome reporter and a Renilla luciferase control. Luciferase activity was analyzed as described above. (E to G) The mutants of huANP32A or swANP32A were co-transfected together with plasmids carrying the polymerases from H7N9 ZJ13 ( E ), H9N2 ZJ12 ( F ), or WSN ( G ). Luciferase activity was analyzed as described above. (B to G, data are Firefly activity normalized to Renilla . Statistical differences between cells are labeled according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: This experiment was carried out using the following primary antibodies: Influenza A Virus PA antibody (NBP2-42874, NOVUS), Influenza A Virus PB1 antibody (NBP2-42877, NOVUS), Influenza A Virus PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).

Techniques: Construct, Transfection, Expressing, Luciferase, Control, Plasmid Preparation, Lysis, Activity Assay, Labeling

Journal: PLoS Pathogens

Article Title: A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs

doi: 10.1371/journal.ppat.1008330

Figure Lengend Snippet: DKO cells were transfected with different ANP32A or mutants or empty vector (0.6μg), and plasmids carrying components of the viral polymerase (0.6μg PA, 1μg PB1, and 1μg PB2) from avian influenza virus H7N9 ZJ13 (A) , H9N2 ZJ12 (B) , or human influenza virus WSN (C) . The cells were lysed at 24 h post-transfection. Co-IP was performed using Anti-FLAG M2 Magnetic Beads, followed by Western blotting to detect the ANP32A and viral proteins by using specific antibodies, PA antibody (NBP2-42874, NOVUS), PB1 antibody (NBP2-42877, NOVUS), PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).

Article Snippet: This experiment was carried out using the following primary antibodies: Influenza A Virus PA antibody (NBP2-42874, NOVUS), Influenza A Virus PB1 antibody (NBP2-42877, NOVUS), Influenza A Virus PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).

Techniques: Transfection, Plasmid Preparation, Virus, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Strains used for phylogenetic analyses of Aspergillus section Circumdati .

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: Infection

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Mycotoxins produced in section Circumdati (*++: strong production, +: medium production, w (weak): trace production, −: toxin not detected).

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: Produced

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Presence–absence data for extrolites produced in Aspergillus section Circumdati .

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: Produced

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Growth rate comparison of Aspergillus section Circumdati species after 7 d (in mm).

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: Comparison

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Most important colony characters for species recognition in Aspergillus section Circumdati .

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: Cream

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Most important colony characters for species recognition in Aspergillus section Circumdati .

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: Produced

Journal: Studies in Mycology

Article Title: Ochratoxin production and taxonomy of the yellow aspergilli ( Aspergillus section Circumdati )

doi: 10.1016/j.simyco.2014.07.001

Figure Lengend Snippet: Most important micromorphological characters for species recognition in Aspergillus section Circumdati .

Article Snippet: A. robustus , CBS 428.77T = NRRL 6362 = ATCC 36106 = IMI 216610 = NRRL A-17351 = WB 5286 , Surface soil from thorn-forest, near Mombasa, Kenya , EF661176 , EU014101 , EF661357.

Techniques: