† anti human notch2 intracellular domain polyclonal antibody  (R&D Systems)

Journal: Scientific Reports

Article Title: Notch signaling drives development of Barrett’s metaplasia from Dclk1-positive epithelial tuft cells in the murine gastric mucosa

doi: 10.1038/s41598-021-84011-4

Figure Lengend Snippet: Metaplastic lesions in pL2.Dclk1.N2IC mice are partially derived from Dclk1-positive gastric tuft cells. ( A ) Representative stainings for Notch2-IC of the SCJ of pL2.Dclk1 and pL2.Dclk1.N2IC mice, respectively; scale bars = 200 µm; insets scale bar = 50 µm. ( B ) Histopathologic scoring of Notch2-IC stained nuclei in pL2.Dclk1.N2IC tissues compared to pL2.Dclk1 controls (6 + 3 n = 25 pL2.Dclk1.N2IC, n = 11 pL2.Dclk1 controls; ordinary two-way ANOVA, p ≤ 0.0001; 6 + 6 n = 20 pL2.Dclk1.N2IC, n = 7 pL2.Dclk1 controls; ordinary two-way ANOVA, p = 0.046). ( C ) Survival analysis of pL2.Dclk1.N2IC compared to pL2.Dclk1 controls shows significant decrease of median overall survival in pL2.Dclk1.N2IC mice (n = 41 pL2.Dclk1.N2IC; n = 31 pL2.Dclk1 controls; Median survival pL2.Dclk1.N2IC = 3.3 months; Median survival pL2.Dclk1 ≥ 10 months; log-rank (Mantel-Cox) test, Chi square = 25,91, p ≤ 0.0001). ( D , E ) Histopathologic analysis and scoring in pL2.Dclk1.N2IC tissues compared to pL2.Dclk1 controls for PAS-positive cells at the SCJ (6 + 3 n = 21 pL2.Dclk1.N2IC, n = 6 pL2.Dclk1 controls; ordinary two-way ANOVA, p = 0.025; 6 + 6 n = 9 pL2.Dclk1.N2IC, n = 2 pL2.Dclk1 controls) and mucous producing cells (6 + 3 n = 21 pL2.Dclk1.N2IC, n = 6 pL2.Dclk1 controls; 6 + 6 n = 6 pL2.Dclk1.N2IC, n = 4 pL2.Dclk1 controls, ordinary two-way ANOVA, p = 0.049); scale bars = 200 µm; error bars indicate SEM.

Article Snippet: Standard immunohistochemical procedures with citrate buffer antigen retrieval (1.00244.1000, Merck) were performed using following antibodies: primary rat anti-human Notch2-IC antibody (DSHB Hybridoma Bank, C651.6DbHN-c, 1:500; overnight at 4 °C), rabbit anti-mouse DCLK1 antibody (Abgent, AP7219b), rabbit anti-mouse c-Myc (Abcam, ab32072), mouse anti-mouse β-Catenin (BD Biosciences, 610154), and secondary anti-rat (Vector Labs, BA-4000), anti-rabbit (Vector Labs, BA-1000) and anti-mouse (Vector Labs, BA-9200) antibody.

Techniques: Derivative Assay, Staining

Journal: Scientific Reports

Article Title: Notch signaling drives development of Barrett’s metaplasia from Dclk1-positive epithelial tuft cells in the murine gastric mucosa

doi: 10.1038/s41598-021-84011-4

Figure Lengend Snippet: Genetic ablation of the Notch 2 receptor in Dclk1-positive gastric tuft cells decelerates BE progression. ( A ) Experimental scheme illustrating timepoints of Tamoxifen induction and tissue collection for analysis. ( B ) Representative stainings for Notch2-IC of the SCJ of pL2.Dclk1 and pL2.Dclk1.N2fl mice, respectively; scale bars = 200 µm; insets scale bar = 50 µm; for pL2.Dclk1, at 6 + 3 and 6 + 6 the same pictures are shown as in Fig. A for comparison to pL2.Dclk1.N2fl tissues. ( C ) Histopathologic scoring of Notch2-IC stained nuclei in pL2.Dclk1.N2fl tissues compared to pL2.Dclk1 controls (6 + 3 n = 10 pL2.Dclk1.N2fl, n = 11 pL2.Dclk1 controls; 6 + 6 n = 15 pL2.Dclk1.N2fl, n = 7 pL2.Dclk1 controls; 6 + 9 n = 12 pL2.Dclk1.N2fl, n = 14 pL2.Dclk1 controls, ordinary two-way ANOVA, p = 0.014). ( D , E ) Histopathologic analysis and scoring in pL2.Dclk1.N2fl tissues compared to pL2.Dclk1 controls for PAS-positive cells at the SCJ (6 + 3 n = 6 pL2.Dclk1.N2fl, n = 6 pL2.Dclk1 controls, ordinary two-way ANOVA, p = 0.017; 6 + 6 n = 9 pL2.Dclk1.N2fl, n = 2 pL2.Dclk1 controls; 6 + 9 n = 5 pL2.Dclk1.N2fl, n = 7 pL2.Dclk1 controls, ordinary two-way ANOVA, p = 0.0009) and mucous producing cells (6 + 3 n = 5 pL2.Dclk1.N2fl, n = 6 pL2.Dclk1 controls, ordinary two-way ANOVA, p = 0.0057; 6 + 6 n = 9 pL2.Dclk1.N2fl, n = 4 pL2.Dclk1 controls; 6 + 9 n = 7 pL2.Dclk1.N2fl, n = 7 pL2.Dclk1 controls); scale bars = 200 µm; insets scale bar = 50 µm; error bars indicate SEM.

Article Snippet: Standard immunohistochemical procedures with citrate buffer antigen retrieval (1.00244.1000, Merck) were performed using following antibodies: primary rat anti-human Notch2-IC antibody (DSHB Hybridoma Bank, C651.6DbHN-c, 1:500; overnight at 4 °C), rabbit anti-mouse DCLK1 antibody (Abgent, AP7219b), rabbit anti-mouse c-Myc (Abcam, ab32072), mouse anti-mouse β-Catenin (BD Biosciences, 610154), and secondary anti-rat (Vector Labs, BA-4000), anti-rabbit (Vector Labs, BA-1000) and anti-mouse (Vector Labs, BA-9200) antibody.

Techniques: Comparison, Staining

Journal: BMC veterinary research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells.

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: Fig. 6 a Western blot analysis using anti-human Notch2 antibody. HeLa cell line was used as a positive control. b Notch2 protein expression levels in HSA cell lines cultured under the normal or serum-free condition, c Conceptual diagram of Notch2 protein and its mutants. d Gene expression levels of Notch target genes. Vec = cells transfected the empty vector. FL = cells overexpressing full length of Notch2. Ex = cells overexpressing dominant negative form of Notch2. In = cells overexpressing constitutive active form of Notch2. Gene expression levels of Vec were set to 1. *p < 0.01. **p < 0.05. Dunnett’s test. Samples for gene expression analysis were analyzed in triplicates and the scores are presented as means ± SD

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Western Blot, Positive Control, Expressing, Cell Culture, Gene Expression, Transfection, Plasmid Preparation, Dominant Negative Mutation

Journal: BMC veterinary research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells.

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: Fig. 7 a Colony numbers of HSA cell lines overexpressing Notch2 vector constructs. b (Top) Representative images of JuB2 and Ud6, 5 and 7 days after starting serum-free culture, respectively. Bars = 100 μm. (Bottom) Relative cell numbers of JuB2 and Ud6. Number of Vec was set to 1. *p < 0.01. **p < 0.05. Dunnett’s test. The scores are presented as means ± SD

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Plasmid Preparation, Construct

Journal: BMC veterinary research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells.

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: Fig. 8 Immunohistochemistry analysis for clinical HSA cases using anti- human Notch2 antibody. Insertion indicated the magnified views of tumor cells or normal endothelial cells. Arrows = neoplastic cells. Arrow heads = endothelial cells in normal blood vessels in the same slides. Asterisks = lymphocytes. Bars = 50 μm

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Immunohistochemistry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Notch in activation and effector functions of γδ T cells.

doi: 10.4049/jimmunol.1300369

Figure Lengend Snippet: FIGURE 1. Notch receptors are expressed on peripheral gd T cells. gd T cells were MACS purified from the peripheral blood. (A) Real-time quantitative PCR showed mRNA expression of Notch1, Notch2, Dll1, and Jag1 on gd T cells. Data were normalized to expression of GAPDH. Data represent mean of four independent experiments. (B) Expression of N1ICD, Notch2 intracellular domain, Dll1, and Jag1 on gd T cells. The MFI in each case has been corrected for the MFI of the isotype control. (C) Purified gd T cells were negative for CD14, CD15, CD19, and CD33 receptors, and were positive for CD56 marker. Filled histogram indicates the isotype control.

Article Snippet: Cells were stained with allophycocyaninlabeled mouse anti-human gd TCR Ab (BD Bioscience, San Diego, CA), sheep anti-human N1ICD Ab, or goat anti-human Notch2 intracellular domain Ab (R&D Systems) for 45 min at 4 ̊C.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Notch in activation and effector functions of γδ T cells.

doi: 10.4049/jimmunol.1300369

Figure Lengend Snippet: FIGURE 5. Notch signaling regulates cytolytic effector functions of gd T cells. (A) Expression of CD107a or LAMP1 (marker for degranulation) on gd T cells. Effector gd T cells alone or in the presence of rIL-2 were cocultured with target oral cancer cell line (AW13516) at the ratio of 4:1 and incubated for 4 h. AW13516 were previously treated with zoledronate (100 mM) for 16 h. Decreased expression of CD107a was observed when the cells were treated with GSI-X (lower panel). Panel is representative of three experiments. (B) Zoledronate-treated AW13516 cells labeled with [51Cr] used as target cells were cocultured with gd T cells at different E/T ratios in a standard 4-h [51Cr] release assay. Data represent percentage cytotoxicity as described in Materials and Methods. Representative data of an independent experiment. (C) Cytolytic ability of gd T cells alone or in the presence of rIL-2 against zoledronate-treated AW13516 was determined by [51Cr] release assay at the ratio of 40:1. Treatment of gd T cells with GSI-X blocked the target cell lysis. *p , 0.05 compared with percentage cytotoxicity of both gd T cells and gd T cells with rIL-2 without GSI-X. Results are mean percentage cytotoxicity of gd T cells from five healthy individuals. (D) gd cells were transfected with 50 nM control (SiGLO) or different Notch-specific SiRNA duplexes. Forty-eight hours post- transfection, cells were harvested and samples were subjected to Western blot analysis for Notch1 and Notch2 (top panel) or b-Actin (bottom panel). The latter was included as loading/normalization control. (E) gd T cells were transfected with 50 nM control (SiGLO) or specific Notch1 and Notch2 siRNA duplexes, Forty-eight hours posttransfection, the cytolytic effector function of rIL-2–activated gd T cells against zoledronate-treated AW13516 was de- termined by [51Cr] release assay at the ratio of 40:1. Silencing of either Notch1 or Notch2 inhibits target cell lysis by gd T cells. **p , 0.005 compared with percentage cytotoxicity of Notch-specific siRNA with SiGLO-treated gd T cells. The results are mean percentage cytotoxicity of gd T cells from five healthy individuals.

Article Snippet: Cells were stained with allophycocyaninlabeled mouse anti-human gd TCR Ab (BD Bioscience, San Diego, CA), sheep anti-human N1ICD Ab, or goat anti-human Notch2 intracellular domain Ab (R&D Systems) for 45 min at 4 ̊C.

Techniques: Expressing, Marker, Incubation, Labeling, Release Assay, Lysis, Transfection, Control, Western Blot