mouse anti pedv nucleoprotein (n) monoclonal antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

doi: 10.3389/fimmu.2024.1451154

Figure Lengend Snippet: Porcine enteroids on transwell culture (PETCs) derived from three regions (duodenum, jejunum, and ileum) of porcine small intestine inoculated with PEDV and mock. Representative images of PETCs inoculated with porcine epidemic diarrhea virus (PEDV, USA/Colorado/2013) at 1.58 x 10 5 TCID50/mL or mock-inoculated and incubated for 24 h (n=4); Bright-field images of PEDV-inoculated cells showing cytopathic changes such as increased detachment of cells (A–C) as compared to the mock-inoculated (D–F) . Immunofluorescence images showing for PEDV nucleocapsid (N) protein (green) following staining with an IgG1 fluorescein isothiocyanate (FITC)-conjugated anti-PEDV N protein monoclonal antibody (Medgene) at 1:100 dilution in PBS pH 7.4 with 0.1% bovine serum albumin (BSA; Jackson Immuno Research). Intense green fluorescent signals can be observed in the infected PETCs (G–I) as compared to the mock-inoculated (J–L) . (M) Bar graph showing the trend of integrated fluorescence intensity values (y-axis) obtained from Image J analysis of 200X magnified images representative of two experiments. The product of the mean fluorescence intensity and the percentage of area with fluorescent signal was termed integrated fluorescence intensity. (N) Bar graph representing normalized gene expression of PEDV N-gene against EIF3K endogenous control gene in PETCs derived from different small intestinal segments. The relative quantification values (y-axis) data obtained from ΔΔCt analysis showed an average of four replicates (Error bars = SEM) with P value < 0.001 (***) denoting statistical significance, while ns denotes no significance.

Article Snippet: After washing twice each well with 200 μL PBS, 100 μL of fluorescein isothiocyanate (FITC)-conjugated mouse anti-PEDV nucleocapsid (N) protein IgG1 monoclonal antibody (Medgene, Brookings, SD, USA) diluted 1:100 in PBS pH 7.4 containing 0.1% bovine serum albumin (BSA; Jackson Immuno Research, West Grove, PA, USA) was added per well and incubated for 1 h at 37°C.

Techniques: Derivative Assay, Virus, Incubation, Immunofluorescence, Staining, Infection, Fluorescence, Gene Expression, Control, Quantitative Proteomics

Journal: Virologica Sinica

Article Title: Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response

doi: 10.1016/j.virs.2024.05.006

Figure Lengend Snippet: Effect of knockdown and overexpression of CALML5 on PEDV infection. A – C Vero E6 cells were transfected with siRNA targeting CALML5 (siCALML5) or negative control (siNC), and then infected with PEDV (CV777) at an MOI of 1. Cells were harvested at pointed times. The protein level was analyzed by Western blotting (WB) ( A ), virus titer was determined using TCID 50 with the method of Reed-Muench ( B ), and the mRNA level of PEDV-N was analyzed by RT-qPCR ( C ). D CALML5-knockout Vero cells and wild-type Vero cells were infected with 0.1 MOI PEDV (CV777), and then cells were harvested at pointed times. The protein level was analyzed by WB. E , F Vero cells were transfected with empty vector pFLAG or recombinant plasmid pFLAG-tagged CALML5. At 24 ​h post-transfection, cells were infected with PEDV (CV777) at an MOI of 0.1, and cells were harvested for indicated times. The protein level was analyzed by WB ( E ). Virus titer was determined using TCID 50 with the method of Reed-Muench ( F ). Data presented as three independent experiments (means ​± ​SD). Statistical analyses were performed using one-way analysis of variance. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ns showed no significant difference.

Article Snippet: Mouse monoclonal anti-PEDV-N was from Alpha Diagnostic International (San Antonio, TX, USA).

Techniques: Knockdown, Over Expression, Infection, Transfection, Negative Control, Western Blot, Virus, Quantitative RT-PCR, Knock-Out, Plasmid Preparation, Recombinant

Journal: Virologica Sinica

Article Title: Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response

doi: 10.1016/j.virs.2024.05.006

Figure Lengend Snippet: CALML5 is involved in viral internalization of PEDV. A , B CALML5 participates in PEDV internalization instead of viral attachment. Vero E6 cells were transfected with siCALML5 or siNC, and then infected with PEDV (CV777) at an MOI of 5 ​at 4 ​°C for 1 ​h. In viral attachment test ( A ), the infected cells were harvested after washing with PBS. In viral internalization test ( B ), after washing with PBS, cells were incubated at 37 ​°C for another 1 ​h. Then the infected cells were washed with PBS and harvested. The mRNA levels of PEDV-N analyzed by RT-qPCR. C Vero cells were pre-treated with rabbit antibody against CALML5 (diluted at 1:100) and DR5 (1:100) or rabbit IgG (1:100) for 2 ​h. After washing with PBS, cells were infected with PEDV (MOI of 5) at 37 ​°C for 2 ​h. The mRNA levels of PEDV-N analyzed by RT-qPCR. D CALML5 is also involved in PEDV release. Vero E6 cells transfected with siCALML5 or siNC and then infected with PEDV (CV777) at MOI of 0.5. PEDV titers in cells (intracellular) and culture fluid (extracellular) were determined by TCID 50 assay at different infection times. E Vero E6 cells were transfected with siCALML5 or siNC and then infected with PEDV at MOI of 0.5 for different times, cell samples were collected, and the levels of negative-strand viral RNA (in the presence of CHX at 2 hpi) were analyzed by RT-qPCR. Data presented as three independent experiments (means ​± ​SD). Statistical analyses were performed using one-way analysis of variance. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ns showed no significant difference.

Article Snippet: Mouse monoclonal anti-PEDV-N was from Alpha Diagnostic International (San Antonio, TX, USA).

Techniques: Transfection, Infection, Incubation, Quantitative RT-PCR

Journal: Virologica Sinica

Article Title: Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response

doi: 10.1016/j.virs.2024.05.006

Figure Lengend Snippet: CALML5 regulates late endosome traffic by targeting ESCRT. A Vero E6 cells were transfected with siCALML5 or siNC, and mRNA levels of ESCRT components were analyzed by RT-qPCR. B Quantitative analysis in mRNA level of ESCRT components. Vero E6 cells were transfected with siCALML5 or siNC, and then mock infected or infected with PEDV at MOI of 1 for indicated times. The mRNA level of ESCRT components was analyzed by RT-qPCR. C The effect of knockdown of ESCRT components on PEDV replication. Vero cells were transfected with siTSG101, siVPS28, siMVB12, siEAP20 or siNC, and then infected with PEDV (CV777) at MOI of 1 for 12 ​h (left) or 24 ​h (right). The PEDV-NP level was quantified by Western blotting (WB) and the intensity band ratio of PEDV-NP to GAPDH was analyzed by using ImageJ software ( B ). Under the same experimental conditions, titers of viruses were measured, as log 10 ​PFU/mL ( C ). D – F A screened ESCRT components participate in viral internalization of PEDV. Vero E6 cells were transfected with siTSG101, siVPS28, siMVB12, siEAP20 or siNC, and infected with PEDV (CV777) at an MOI of 5 ​at 4 ​°C for 1 ​h. In viral attachment test ( D , left), the infected cells were harvested after washing with PBS. In viral internalization test ( D , right), after washing with PBS, cells were incubated at 37 ​°C for another 1 ​h. Then the infected cells were washed with PBS and harvested. The mRNA levels of PEDV-N were analyzed by RT-qPCR. E A screened ESCRT components are involved in PEDV release. Vero cells transfected with siTSG101, siVPS28, siMVB12, siEAP20 or siNC, and then infected with PEDV at MOI of 0.5 for 12 ​h. PEDV titers in cells (intracellular) and culture fluid (extracellular) were determined by TCID 50 assay at different times. F , G Cytotoxicity of the siRNAs and knockdown efficiency. The Vero cells grown in 96-well plates were transfected with the indicated siRNAs, and the cell viability was evaluated by using CCK8 method ( F ). Total RNA was extracted and mRNA level was analyzed by RT-qPCR ( G ). The data were presented as mean ​± ​SEM of three independent experiments. Statistical analyses were performed using one-way analysis of variance. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ns showed no significant difference.

Article Snippet: Mouse monoclonal anti-PEDV-N was from Alpha Diagnostic International (San Antonio, TX, USA).

Techniques: Transfection, Quantitative RT-PCR, Infection, Knockdown, Western Blot, Software, Incubation