Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) PCR amplification results after five blind passages. ( B ) TEM observation of CPV L4. Arrows indicate spherical virus particles; scale bar, 50 nm. ( C ) Cytopathic effects of CPV L4 infection in F81 cells (MOI = 1). ( D ) Western blot analysis of VP2 expression in CPV L4-infected F81 cells (MOI = 1). Upper band was observed above the VP2 protein band, which may be caused by post-translational modification of the VP2 protein or incomplete denaturation. GAPDH served as an internal control. ( E ) Immunofluorescence detection of CPV L4-infected F81 cells (MOI = 1); scale bar, 100 µm. ( F ) Growth kinetics of CPV L4 in F81 cells (MOI = 0.1). Infected cells and supernatants were harvested every 12 h for 72 h. Viral genome copies are shown as log10(copies/µL). Data represent three independent experiments (triplicate each) and are presented as mean ± SEM.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Amplification, Virus, Infection, Western Blot, Expressing, Modification, Control, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: Genetic and evolutionary analysis of CPV VP2. ( A ) Maximum-likelihood phylogenetic tree of the CPV VP2 gene constructed using MEGA X with the JTT+G substitution model and 1000 bootstrap replicates. Orange circles indicate isolates CPV L1–L8. ( B ) Heatmap of amino acid similarity among CPV VP2 proteins. ( C ) VP2 amino acid mutation analysis of isolates CPV L1–L8 relative to prototype CPV-2. ( D ) Visual alignment of partial VP2 sequences between CPV L4 and giant panda-derived CPV-2c strains. The orange arrow indicates the full-length VP2 sequence of CPV L4, and blue arrows indicate reported partial VP2 sequences of four panda-infecting CPV-2c strains. Sequences are aligned by VP2 amino acid positions.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Construct, Mutagenesis, Derivative Assay, Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Schematic of nanobody expression construct. Nanobody genes are expressed under the Pgrac promoter, fused with the amyQ signal peptide for secretion via the Sec pathway, and linked to a 6 × His tag for detection and purification. ( B ) SDS-PAGE analysis of five nanobodies in Bacillus subtilis culture supernatants. ( C ) Western blot identification of His tags for five nanobodies. ( D ) Indirect ELISA evaluation of binding reactivity between nanobodies and immunization VP2 protein; results are shown as OD450 values. * p < 0.05, **** p < 0.0001.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Expressing, Construct, Purification, SDS Page, Western Blot, Indirect ELISA, Binding Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Western blot validation of Nb10 recognition of VP2 in CPV L4-infected cells (MOI = 1); Nb10 served as the primary antibody. ( B ) Immunofluorescence detection of Nb10 recognition at the cellular level (MOI = 1); Nb10 served as the primary antibody; nuclei were stained with DAPI (blue). ( C ) Molecular docking model of Nb10 with CPV VP2 showing potential interaction sites. ( D ) Visualization of the Nb10–VP2 complex from different angles.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Western Blot, Biomarker Discovery, Infection, Immunofluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Schematic of Nb10-Fc expression construct. ( B ) Western blot validation of Nb10-Fc expression. Nb10-Fc’ indicates the non-reducing condition result; Nb10-Fc indicates the reducing condition result. Nb10-Fc was detected under both conditions but showed different migration characteristics. ( C ) Indirect ELISA evaluation of Nb10-Fc binding to immunization VP2 protein (OD450). ( D ) Western blot validation of Nb10-Fc recognition of VP2 in CPV L4-infected cells (MOI = 1). Upper band was observed above the VP2 protein band, which may be caused by post-translational modification of the VP2 protein or incomplete denaturation. GAPDH served as an internal control. ( E ) Immunofluorescence detection of Nb10-Fc recognition in CPV L4-infected cells (MOI = 1). Specific green fluorescence was observed in infected cells; nuclei were stained with DAPI (blue). ( F ) Molecular docking model and interface analysis of Nb10-Fc with CPV VP2. ( G ) Visualization of the Nb10-Fc–VP2 complex from different angles. **** p < 0.0001.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Expressing, Construct, Western Blot, Biomarker Discovery, Migration, Indirect ELISA, Binding Assay, Infection, Modification, Control, Immunofluorescence, Fluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: Dynamic property analyses of CPV L4 VP2–Nb10 (left) and CPV L4 VP2–Nb10-Fc (right) complexes. ( A ) RMSD curves; ( B ) RMSF curves (green shaded regions indicate interaction interfaces); ( C ) hydrogen bond numbers; ( D ) SASA curves; ( E ) radius of gyration (Rg) curves.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques:

Journal: PLOS One

Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

doi: 10.1371/journal.pone.0345169

Figure Lengend Snippet: A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, β-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).

Article Snippet: Primary mouse anti-β-actin mAb (Santa Cruz Biotechnology, SC-69679) and IRdye-conjugated donkey anti-mouse IgG (LI-COR, Lincoln, Neb) were used as a loading control.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Staining, Transduction, Microscopy, Fluorescence, shRNA, Infection