Journal: Scientific Reports

Article Title: HIF-PHD inhibitor regulates the function of group2 innate lymphoid cells and polarization of M2 macrophages

doi: 10.1038/s41598-023-29161-3

Figure Lengend Snippet: IL-33-ST2L-p38 MAPK signaling was impeded in kidney ILC2s treated with HIF-PHD inhibitors. ( A ) ILC2s were sorted from kidneys of pooled 6 mice, and cultured with IL-2 and IL-7 for 3 days. Then IL-33 was added to stimulate ILC2s for further 3 days, and HIF-PHD inhibitors were added for the last 24 h. Relative levels of mRNA expression for ST2L in kidney ILC2s treated with DMSO, GSK360A, and FG-4592. ( B ) Sorted kidney ILC2s were stimulated by 50 ng/ml recombinant murine IL-33 or 50 ng/ml PMA and 500 ng/ml ionomycin for 3 h in the presence of brefeldin A. ST2L MFI was analyzed by FACS. ( C ) Sorted kidney ILC2s were cultured with IL-2 and IL-7 for 3 days, and half of the media was changed with fresh media containing IL-2 and IL-7. After a further 3 days, ILC2s were cultured in cytokine-free conditions for 4 h in the presence of HIF-PHD inhibitors, and then ILC2s were stimulated with 10 ng/ml recombinant murine IL-33 for 15 min. Phosphorylated p38 MAPK (Thr180/Tyr182) was assessed by FACS. Representative histograms are shown for phosphorylated p38 MAPK in kidney ILC2s treated with DMSO (gray area), GSK360A (dashed line), FG-4592 (long-dashed line), IL-33 non-stimulated (solid line), and isotype control (black area). ( D ) The graph shows the frequency of phosphorylated p38 MAPK. We used the pooled kidney from 6 mice as n = 1, and data shown are pooled from two (A,B) (n = 6) or three (D) (n = 9) independent experiments. Error bars show SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To detect phosphorylated-p38 MAPK, ILC2s were cultured with cytokine free condition for 4 h, and stimulated with 10 ng/ml IL-33 for 15 min, and immediately fixed/permeabilized by pre-chilled methanol, and stained with anti-p38 MAPK Phospho (Thr180/Tyr182) antibody (Biolegend).

Techniques: Cell Culture, Expressing, Recombinant, Control

Journal: Frontiers in Immunology

Article Title: Impact of Enhancer of Zeste Homolog 2 on T Helper Cell-Mediated Allergic Rhinitis

doi: 10.3389/fimmu.2017.00790

Figure Lengend Snippet: House dust mite-induced enhancer of zeste homolog 2 (Ezh2)-mediated T-lymphocyte response was modified by p38 inhibitor. (A) Western blot analysis of Ezh2, p38, pp38, and Actin in Jurkat cells after adding p38 MAP kinase inhibitor, * p < 0.05, by the t -test. (B) Relative Ezh2 expression of Th1 cells to naïve CD4 + T cells with HDM stimulation with or without p38 MAP kinase inhibitor treatment on day 10. The mean fluorescence intensity (MFI) of Th1 cells divided by the MFI of naïve CD4 + T cells in the same tube [(MFI of Th1 cells/MFI of naïve CD4 + T cells) × 100%] ( n = 11). (C) The mean fold change of Th1 percentage in CD4 + T cells with HDM stimulation with or without p38 MAP kinase inhibitor treatment on day 10 compared to day 0 (the percentage on day 0/the percentage on day 10) ( n = 11). (D) Relative Ezh2 expression of Th2 cells to naïve CD4 + T cells with HDM stimulation with or without p38 MAP kinase inhibitor treatment on day 10. The MFI of Th1 cells divided by the MFI of naïve CD4 + T cells in the same tube [(MFI of Th2 cells/MFI of naïve CD4 + T cells) × 100%] ( n = 11). (E) The mean fold change of Th2 percentage in CD4 + T cells with HDM stimulation with or without p38 MAP kinase inhibitor treatment on day 10 compared to day 0 (the percentage on day 0/the percentage on day 10) ( n = 11 in each group).

Article Snippet: Antibodies used in this study described as follows: anti-p38 antibody (ABGENT, Catalog Number: AP3904a, San Diego, CA, USA); anti-pp38 antibody (Thr180/Tyr82, ABGENT, Catalog Number: AP52412, San Diego, CA, USA); anti-Ezh2 antibody (ABGENT, Catalog Number: AW5436-U400, San Diego, CA, USA); anti-IL4 antibody (PE, Clone: 8D4-8, Catalog Number: 12-7049, eBioscience, San Diego, CA, USA); anti-IFN-γ antibody (PE, Clone: 4S B3, Catalog Number: 12-7319, eBioscience, San Diego, CA, USA); anti-CD4 antibody (PERCP-CY5.5, Clone: RPA-T4, Catalog Number: 45-0049, eBioscience, San Diego, CA, USA); anti-CD45ra antibody (APC, Clone: MP4-25D2, Catalog Number: 555490, BD, San Diego, CA, USA); Donkey anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody (DyLight ® 488, Catalog Number: SA5-10026, eBioscience, San Diego, CA, USA).

Techniques: Modification, Western Blot, Expressing, Fluorescence

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Primers used for quantitative RT-qPCR analysis.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Sequencing

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Oligos and primers used for making gRNA plasmids or PCR identification of pp38-deleted MDV mutants.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Sequencing

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: PCR analysis and sequence alignment of the double-strand breaks in pp38-deleted MDV mutants: ( a ) PCR analysis of the mutated or wild-type pp38 genes of GX0101 edited by crossed combinations of different gRNAs. ( b ) Sequence alignment of double-strand breaks (DSBs) in pp38 genes mutated by the gRNA combination gRF2/gRR1. ( c , d ) PCR analysis of pp38 deletions in single MDV plaques from the first and second rounds of virus purification, respectively. Due to limited space, only some of the results are shown here. The entire or broken gRNA sequences and protospacer-adjacent motifs (PAMs) are shown by same-colored arrows or square frames in red, respectively.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Sequencing, Purification

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: IFA staining and RT-qPCR analysis of the expression of viral genes in MDV-infected CEFs: ( a ) Expression of the MEQ and pp38 proteins in GX0101 or GX0101∆pp38-infected CEFs, as detected by immunofluorescence assay (scale bar = 50 µm). ( b ) Relative expression levels of MDV-1 genes in GX0101- or GX0101∆pp38-infected CEFs, as determined by RT-qPCR analysis. All of the experiments were independently repeated three times, and the gene expression levels were normalized to that of the MDV oncogene meq. Asterisks (*) indicate statistically significant differences between the pp38-deleted mutant and the parental GX0101; **, p < 0.01.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Staining, Quantitative RT-PCR, Expressing, Infection, Immunofluorescence, Mutagenesis

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: List of the hybridomas secreting MDV-specific monoclonal antibodies.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques:

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Cross-screening of pp38-specific monoclonal antibodies by immunofluorescence assay. Plaques produced by infection of CEFs with GX0101 or GX0101∆pp38 were stained with 4 different antibodies and visualized by immunofluorescence and bright-field microscopy. IFA, immunofluorescence assay; Merge, fused image. Scale bar = 50 µm.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Immunofluorescence, Produced, Infection, Staining, Microscopy

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Staining of pp38 proteins overexpressed in 293T cells by immunofluorescence assay. The pp38 mAb BD1 served as a positive control. EGFP, enhanced green fluorescent protein with auto-fluorescence in green; pp38, MDV-1 phosphoprotein 38 stained in red; Merge, fused image in yellow. Scale bar = 50 µm.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Staining, Immunofluorescence, Positive Control, Fluorescence

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Confocal analysis of pp38 and gB proteins in MDV-infected CEFs. pp38, MDV-1 phosphoprotein 38 stained in red; gB, MDV-1 glycoprotein B stained in green; DAPI, 4′,6-diamidino-2-phenylindole used to indicate the nuclei of CEFs in blue; Merge, fused image in yellow. Scale bar = 20 µm.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Infection, Staining

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Reactivity of the mAb 31G7 to different serotypes of MDVs: ( a ) Immunofluorescence assays performed for the detection of the reaction spectrum of the pp38 mAb 31G7 to different serotypes of MDVs. Normal images of GX0101 or GX0101∆pp38 plaques under regular light; IFA, immunofluorescence assay. ( b ) Western blot analysis performed for the detection of the reaction spectrum of the mAb 31G7 to proteins from CEFs infected with different serotypes of MDVs. Chicken β-actin was used as the protein loading control. Scale bar = 50 µm.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Immunofluorescence, Western Blot, Infection

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Immunoprecipitation and mass spectrometry analysis of the proteins recognized by the pp38 mAb 31G7: ( a ) Image of the immunoprecipitation and silver-stained SDS gel showing obvious protein bands captured by the pp38 mAb 31G7. The black arrow indicates the band of a target protein about 38 kD in size. ( b ) Image of total ion chromatography (TIC) for the 38 kD target band analyzed by LC–MS/MS.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Immunoprecipitation, Mass Spectrometry, Staining, SDS-Gel, Ion Chromatography, Liquid Chromatography with Mass Spectroscopy

Journal: Viruses

Article Title: A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

doi: 10.3390/v14092045

Figure Lengend Snippet: Expression of pp38 proteins in the feather follicle epithelium of MDV-infected birds, as determined by immunohistochemistry. The feather follicular samples from Md5-challenged birds at 14 days post-infection were subjected to immunohistochemistry analysis using the pp38 mAb 31G7. The black arrows indicate MDV-expressed pp38 proteins in the feather follicle epithelium. Scale bar = 100 µm.

Article Snippet: Then, the tissue sections were prepared and subjected to immunostaining as described previously [ ], using MDV-1 pp38 specific mAb 31G7 and the DAB Kit (Cowin Biosciences, China).

Techniques: Expressing, Infection, Immunohistochemistry