rabbit anti β2 integrin antibody  (Novus Biologicals)

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 1 Overexpression of β2 integrin or siRNA silencing affect Salmonella infection. A, overexpression of β2 integrin facilitated Salmonella infection in transfected HeLa cells. B and C, flow cytometry measurement. D, CFU assays. E, transfection with siRNA resulted in the downregulation of β2 integrin mRNA in transfected HeLa cells. NT, non-targeting siRNA. F and G, silencing of β2 integrin inhibited Salmonella infection of HeLa cells. Data are mean±SD. Unpaired t-test was used for the statistical analysis, and a P-value<0.05 was considered significant. *, P<0.05; **, P<0.01; ***, P<0.001. ns, not significant. Scale bars=400 µm in A and F.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Over Expression, Infection, Transfection, Flow Cytometry

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 2 β2 integrin is required for Salmonella binding. A, silencing of β2 integrin inhibited Salmonella binding of HeLa cells. HeLa cells were infected with Salmonella at 4˚C for 1 h. Cells were stained with anti-Salmonella antibody (green), actin-specific TRITC- phalloidin (red), and DAPI (blue). Samples were imaged using a Zeiss LSM880 laser-scanning confocal microscope and processed using Zeiss ZEN 2.3 blue edition software. Each image shown represents a set of 24 images. Presented are the combined z-stacks for each infected cell. B, number of Salmonella per cell with silencing of β2 integrin. C, overexpression of β2 integrin increased binding of Salmonella to HeLa cells. The samples were stained described as above. D, number of Salmonella per cell with overexpression of β2 integrin. Data are mean±SD. Statistical significance was determined using the Mann-Whitney test, and a P-value<0.05 was considered significant. ****, P<0.0001. ns, not significant. 63× objective lens.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Binding Assay, Infection, Staining, Microscopy, Software, Over Expression, MANN-WHITNEY

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 3 Antibodies to β2 integrin block Salmonella infection of HeLa, Caco-2, and RAW264.7 cells in a dose-dependent manner. A and B, the mAb against β2 integrin blocked the Salmonella infection of HeLa cells. The isotype IgG2a at the highest concentration was used as control. C and D, the pAb against β2 integrin blocked the Salmonella infection of HeLa cells. The isotype IgG at the highest concentration was used as control. E and F, the mAb against β2 integrin blocked the Salmonella infection of Caco-2 cells. G and H, the mAb against β2 integrin blocked the Salmonella infection of RAW264.7 cells. Data are mean±SD. One-way ANOVA was used for the statistical analysis, and P-value<0.05 was considered significant. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ns, not significant. Scale bars=200 µm.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Blocking Assay, Infection, Concentration Assay, Control

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 4 The β2 integrin ectodomain soluble protein (β2 integrin-GST) neutralizes the Salmonella infection in HeLa, Caco-2, and RAW264.7 cells. A and B, β2 integrin-GST neutralizes the Salmonella infection in HeLa cells in a dose-dependent manner. C and D, β2 integrin-GST neutralizes the Salmonella infection in Caco-2 cells. E and F, β2 integrin-GST neutralizes the Salmonella infection in RAW264.7 cells. Data are mean±SD. One-way ANOVA was used for the statistical analysis, and P-value<0.05 was considered significant. **, P<0.01; ****, P<0.0001. ns, not significant. Scale bars=200 µm.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Infection

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 5 The β2 integrin ectodomain soluble protein neutralizes Salmonella infection in mice. A, β2 integrin-GST neutralizes the Salmonella infection in mice. The log-rank (Mantel-Cox) test was used to analyze the statistical difference between the survival rates of the challenged mice. B and C, representative images of H&E-stained sections of caecum. D and E, immunohistochemical staining for β2 integrin in caecum of infected mice and uninfected mice (negative control). F, SDS-PAGE analysis of GST pull- down assay. G, some differentially expressed proteins based on mass spectrometry analysis. H, immunoelectron microscopy analysis for YrbD localization at both OM and IM of Salmonella. SL1344 was immunolabelled for YrbD with 5-nm gold particles. Black squares represent the labeling of YrbD on the OM of Salmonella. Red squares represent the labeling of YrbD on the IM of Salmonella. The zoom-in windows of the positive labeling were provided (right). Black arrows and red arrows represent the location of the OM and IM, respectively. Scale bars=200 nm.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Infection, Staining, Immunohistochemical staining, Negative Control, SDS Page, Pull Down Assay, Mass Spectrometry, Immuno-Electron Microscopy, Labeling

Journal: Journal of Integrative Agriculture

Article Title: Salmonella YrbD protein mediates invasion into the host by interacting with β2 integrin1

doi: 10.1016/j.jia.2023.12.035

Figure Lengend Snippet: Fig. 6 Interactions between β2 integrin and Salmonella YrbD. A, the β2 integrin-Myc interacted with YrbD-Flag in Co-IP assays with plasmid-transfected HEK293 cell lysates. B, purified β2 integrin-GST pulled down YrbD-Flag. C, purified β2 integrin-GST pulled down purified YrbD-His. D, the extracellular domain of β2 integrin interacted with YrbD-Flag. E and F, deletion of β2 integrin I-like domain abolished the interaction between YrbD and β2 integrin.

Article Snippet: For immunohistochemistry, after incubating with 1:50 diluted rabbit anti-β2 integrin antibody (NBP1-88127, Novus, St. Charles, MO, USA) overnight at 4°C, the sections were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at RT, then rinsed thrice with PBS for 5 min each, stained with diaminobenzidine (DAB) (Sigma-Aldrich), and counterstained with hematoxylin.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Purification