Journal: Viruses

Article Title: Zika Virus Antibody Titers Three Years after Confirmed Infection

doi: 10.3390/v13071345

Figure Lengend Snippet: Results from serological assays for ZIKV and DENV antibodies . A : Percentage of positive and negative tested sera with ZIKV VNT and ZIKV NS1 IgG ELISA. B : Correlation between ZIKV NS1 IgG ELISA ratios and titers from the ZIKV VNT. The dotted lines indicate cut-off values for a positive test result. C : ZIKV- and DENV-2 VNT titers from all participants. Lines represent median ± IQR. The dotted line indicates the cut-off value for a positive test result. Statistical differences were tested with the Mann–Whitney test. D : Correlation between DENV-2 VNT titers and ZIKV VNT titers. The dotted lines indicate cut-off values for a positive test result. E : Correlation between ZIKV NS1 IgG ELISA ratios and DENV-2 VNT titers. The dotted lines indicate cut-off values for a positive test result. * P < 0.05.

Article Snippet: Slides were printed with DENV1–4 and ZIKV NS1 proteins (Sino Biological Europe GmbH and Immune Technology Corp., New York, NY) and Equad proteins (DENV envelope proteins containing four amino acid mutations in the highly conserved fusion loop domain to reduce flavivirus cross-reactivity) [ ].

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Viruses

Article Title: Zika Virus Antibody Titers Three Years after Confirmed Infection

doi: 10.3390/v13071345

Figure Lengend Snippet: Heatmap of results from the different serological assays used in this study . A : IgG antibody titers for DENV1–4 Equad and DENV1–4 and ZIKV NS1 antigens determined with a protein microarray. Corresponding ZIKV and DENV-2 VNT titers and ZIKV NS1 IgG ELISA ratios from all participants are shown on the right. Antibody patterns are ranked from highest to lowest ZIKV VNT titer. B : Protein microarray IgG antibody titer patterns for DENV1–4 Equad and DENV1–4 and ZIKV NS1, ZIKV and DENV-2 VNT titers and ZIKV NS1 IgG ELISA ratios from all participants, ranked from highest to lowest ZIKV NS1 ELISA ratio. Numbers on the left Y-axis are the study numbers of the participants in this study. PA; protein microarray, Equad; envelope proteins containing four amino acid mutations in the highly conserved fusion loop domain to reduce flavivirus cross-reactivity, VNT; virus neutralization test.

Article Snippet: Slides were printed with DENV1–4 and ZIKV NS1 proteins (Sino Biological Europe GmbH and Immune Technology Corp., New York, NY) and Equad proteins (DENV envelope proteins containing four amino acid mutations in the highly conserved fusion loop domain to reduce flavivirus cross-reactivity) [ ].

Techniques: Microarray, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: International Journal of Biological Sciences

Article Title: Curcumol Induces Necroptosis of Hepatic Stellate Cells by Targeting KAT8 to Suppress HK2 Lactylation and Promote HUWE1-Dependent Ubiquitination

doi: 10.7150/ijbs.125009

Figure Lengend Snippet: Curcumol promotes HUWE1-dependent ubiquitin-mediated degradation of HK2 by inhibiting KAT8-mediated lactylation at the K346 site. A, D. Western blot analysis of HK2 protein half-life in LX2 cells treated with or without protein synthesis inhibitors in the presence of sodium lactate (10 mM, 24 h), with quantification of band intensity (n = 3). B. Western blot analysis of HK2 protein levels in LX2 cells transfected with LDHA siRNA or control siRNA and treated with chloroquine (CQ, 20 μg/mL), MG132 (10 μM), alone or in combination for 24 h (n = 3). C. Schematic diagram illustrating regulators of HK2 ubiquitination. E. Co-immunoprecipitation (Co-IP) analysis of the interaction between HK2 and the E3 ubiquitin ligase HUWE1. F. Western blot analysis of HK2 protein levels in LX2 cells treated with MG149 (50 μM, 48 h), with or without MG132 (10 μM, 6 h), followed by densitometric quantification (n = 3). G. Western blot analysis of HK2 protein levels in LX2 cells expressing HK2-WT or HK2-K346A after treatment with MG132 (10 μM, 8 h) (n = 3). H. Co-IP analysis of HK2 ubiquitination in LX2 cells with or without HUWE1 siRNA transfection (24 h). I. Co-IP analysis of HK2 ubiquitination in LX2 cells transfected with or without KAT8 siRNA (24 h) and treated with Curcumol (30 μM). J. Co-IP analysis was used to examine the interaction between HK2 and HUWE1 in LX2 cells that were co-transfected with the indicated plasmids and treated with Curcumol (30 μM, 8 h). K. Co-IP analysis of HK2 ubiquitination in LX2 cells expressing HK2-WT or HK2-K346A. L. Immunofluorescence analysis of HK2 (green), KAT8 (red), and HUWE1 (yellow) colocalization in liver tissues from CCl₄-induced fibrotic mice and Curcumol-treated mice (n = 3; scale bar: 50 μm). M. Co-IP analysis was performed on the co-transfected plasmids for 24 hours, and then the interactions between HK2, KAT8, and HUWE1 in LX2 cells treated with different concentrations of Curcumol (0, 20, 30, 45 μM) were examined. N, Q. Western blot analysis of HK2 degradation kinetics in LX2 cells expressing HK2-WT or HK2-K346A following HUWE1 siRNA transfection (24 h) and cycloheximide (CHX, 20 μg/mL) treatment for the indicated times (n = 3). O, P. Western blot analysis of HK2 protein levels in LX2 cells transfected with HUWE1 siRNA (24 h), with densitometric quantification (n = 3). Data are shown as mean ± SD, and statistical differences were analyzed by one-way ANOVA. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The antibodies employed in this study include: RIPK1 (17519-1-AP), RIPK3 (17563-1-AP), P-RIPK1 (66854-1-Ig), MLKL (21066-1-AP), P-MLKL (82090-2-RR), HK2 (22029-1-AP), PKM2 (15822-1-AP), LDHA (19987-1-AP), PFKM (55028-1-AP), HUWE1 (19430-1-AP), Ubiquitin (10201-2-AP), HA (51064-2-AP), His (66005-1-Ig), EP300 (20695-1-AP) and P62 (18420-1-AP) from proteintech; KAT8 (sc-271691) from Santa Cruz Biotechnology; KAT6A from Bioswamp; Collagen I (ab26003), β-actin (ab8226), Tubulin (Ab721), anti-mouse IgG (ab190475), anti-rabbit IgG (ab288151) and LC3B (ab192890) from Abcam; Pan-Kla (AB_2868521) from PTM-Bio laboratory; KAT6B(A17116), P-RIPK3(AP1260), AARS(A15017), and AARS2 (A7826) from Abclonal.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Immunofluorescence

Journal: Journal of Virology

Article Title: Direct Activation of Endothelial Cells by SARS-CoV-2 Nucleocapsid Protein Is Blocked by Simvastatin

doi: 10.1128/JVI.01396-21

Figure Lengend Snippet: SARS-CoV2 nucleocapsid protein (NP) is a potent inducer of human endothelial cell activation. (a) HLMECs were incubated with SARS-CoV2 structural proteins (S, N, and E proteins; 1 μg/ml) and five nonstructural proteins (NSP1, NSP3, NSP5, NSP7, and NSP8; 1 μg/ml) for 8 h. (b) HLMECs were treated with 1 μg/ml of NP or 10 ng/ml of TNF-α for different incubation periods as indicated. (c) HLMECs were incubated with indicated concentrations of NP for 8 h. TNF-α at 10 ng/ml served as a positive control. (d) Different cultured cells, including mouse lung vascular endothelial cells (MECs), A549 cells, 293T cells, HUVECs, HAECs, HCAECs, HDMECs, and HLMECs, were treated with NP (1 μg/ml) for 8 h. The expression of ICAM-1, VCAM-1, and VE-cadherin was detected by Western blotting. β-Actin served as a loading control. (e) HLMECs were treated with PBS, NP (1 μg/ml), TNF-α (10 ng/ml), or lipopolysaccharide (LPS) (1 μg/ml) for 8 h. The total RNA was isolated and qPCR was performed for measuring the mRNA levels of TNF-α, ICAM-1, VCAM-1, MCP-1, and IL-6. (f) HLMECs were treated with PBS, NP (1 μg/ml), or TNF-α (10 ng/ml) for 8 h and cocultured with Zombie Red-labeled THP-1 cells for 1 h. After being washed, the adherent cells were imaged and quantitatively analyzed.

Article Snippet: SARS-CoV-2 NSP1 (97-095), NSP5 (10-116), NSP7 (97-096), and NSP8 (97-097) proteins were obtained from Prosci (Poway, CA).

Techniques: Activation Assay, Incubation, Positive Control, Cell Culture, Expressing, Western Blot, Control, Isolation, Labeling

Journal: Journal of Virology

Article Title: Direct Activation of Endothelial Cells by SARS-CoV-2 Nucleocapsid Protein Is Blocked by Simvastatin

doi: 10.1128/JVI.01396-21

Figure Lengend Snippet: SARS-CoV2 nucleocapsid protein (NP) is a potent inducer of human endothelial cell activation. (a) HLMECs were incubated with SARS-CoV2 structural proteins (S, N, and E proteins; 1 μg/ml) and five nonstructural proteins (NSP1, NSP3, NSP5, NSP7, and NSP8; 1 μg/ml) for 8 h. (b) HLMECs were treated with 1 μg/ml of NP or 10 ng/ml of TNF-α for different incubation periods as indicated. (c) HLMECs were incubated with indicated concentrations of NP for 8 h. TNF-α at 10 ng/ml served as a positive control. (d) Different cultured cells, including mouse lung vascular endothelial cells (MECs), A549 cells, 293T cells, HUVECs, HAECs, HCAECs, HDMECs, and HLMECs, were treated with NP (1 μg/ml) for 8 h. The expression of ICAM-1, VCAM-1, and VE-cadherin was detected by Western blotting. β-Actin served as a loading control. (e) HLMECs were treated with PBS, NP (1 μg/ml), TNF-α (10 ng/ml), or lipopolysaccharide (LPS) (1 μg/ml) for 8 h. The total RNA was isolated and qPCR was performed for measuring the mRNA levels of TNF-α, ICAM-1, VCAM-1, MCP-1, and IL-6. (f) HLMECs were treated with PBS, NP (1 μg/ml), or TNF-α (10 ng/ml) for 8 h and cocultured with Zombie Red-labeled THP-1 cells for 1 h. After being washed, the adherent cells were imaged and quantitatively analyzed.

Article Snippet: SARS-CoV-2 NSP1 (97-095), NSP5 (10-116), NSP7 (97-096), and NSP8 (97-097) proteins were obtained from Prosci (Poway, CA).

Techniques: Activation Assay, Incubation, Positive Control, Cell Culture, Expressing, Western Blot, Control, Isolation, Labeling

Journal: Cell reports

Article Title: Memory B Cell Activation, Broad Anti-influenza Antibodies, and Bystander Activation Revealed by Single-Cell Transcriptomics

doi: 10.1016/j.celrep.2019.12.063

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Non-structural Protein 2 / NS2 , Sino Biological , 40012-VNAE.

Techniques: Recombinant, Multiplex Assay, Cell Isolation, Sequencing, Software

Journal: Cell reports

Article Title: Epstein-Barr virus BNRF1 destabilizes SMC5/6 cohesin complexes to evade its restriction of replication compartments

doi: 10.1016/j.celrep.2022.110411

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-SMC6L1 rabbit monoclonal antibody , Boster Biological Technology , A01554-1.

Techniques: Virus, Recombinant, Magnetic Beads, Protease Inhibitor, Transfection, Sequencing, Modification, Mass Spectrometry, Produced, Conjugation Assay, Purification, Cell Culture, Gel Extraction, Reverse Transcription, SYBR Green Assay, Isolation, Sample Purification, Multiplex Assay, Cell Isolation, Mutagenesis, Software

Journal: Structure (London, England : 1993)

Article Title: Structural insights into the assembly and regulation of 2'-O RNA methylation by SARS-CoV-2 nsp16/nsp10.

doi: 10.1016/j.str.2025.03.009

Figure Lengend Snippet: Figure 3. Putative ligand binding pocket of nsp16 (A) Snapshots of various ligands that bind to the pocket in different nsp16/nsp10 structures: adenosine (PDB: 6WKS),8 m7GDP (PDB: 6WQ3),9

Article Snippet: The following antibodies were used: SARS-CoV-2 nsp10 (ProSci, 9179), SARS-CoV-2 nsp16 (ProSci, 9271), anti-rabbit IgG HRP secondary (Cell Signaling, 7074).

Techniques: Ligand Binding Assay

Journal: Structure (London, England : 1993)

Article Title: Structural insights into the assembly and regulation of 2'-O RNA methylation by SARS-CoV-2 nsp16/nsp10.

doi: 10.1016/j.str.2025.03.009

Figure Lengend Snippet: Figure 5. Sampling oligomeric conforma- tions in solution (A) Coomassie staining of crosslinked nsp16/ nsp10 (±RNA) on SDS-PAGE, with protein bands labeled as 1–6. (B) Immunoblotting with nsp16 antibody shows nsp16 in bands 2, 4–6, and part of wider band 3 (3A). (C) Immunoblotting with nsp10 antibody detects nsp10 in bands 1, 5, 6, and parts of wider band 3 (3B and 3C). Cartoon representations of their olig- omeric states are shown below (blue circle: nsp10, green square: nsp16). (D) Coomassie staining of crosslinked quintuple mutant (±RNA), protein bands labeled as 1–6. (E and F) Immunoblotting with nsp16 and nsp10 antibodies, respectively.

Article Snippet: The following antibodies were used: SARS-CoV-2 nsp10 (ProSci, 9179), SARS-CoV-2 nsp16 (ProSci, 9271), anti-rabbit IgG HRP secondary (Cell Signaling, 7074).

Techniques: Sampling, Staining, SDS Page, Labeling, Western Blot, Mutagenesis

Journal: Structure (London, England : 1993)

Article Title: Structural insights into the assembly and regulation of 2'-O RNA methylation by SARS-CoV-2 nsp16/nsp10.

doi: 10.1016/j.str.2025.03.009

Figure Lengend Snippet: Figure 6. Oligomerization via nsp16-nsp16 interfaces and MP analysis (A) nsp16-nsp16 interface 1 in the current struc- ture. (B) Previously reported interface 2 (PDB: 7JYY). (C) An overlay revealing distinct interfaces on opposite faces of the nsp16/nsp10 heterodimer. (D and E) Mass photometry of the (D) WT apo (gray) and RNA-bound (1:1 M ratio, dark green) and (E) quintuple mutant.

Article Snippet: The following antibodies were used: SARS-CoV-2 nsp10 (ProSci, 9179), SARS-CoV-2 nsp16 (ProSci, 9271), anti-rabbit IgG HRP secondary (Cell Signaling, 7074).

Techniques: Mutagenesis