notch2 blocking antibody  (Novus Biologicals)

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: Expression of Notch2 in first trimester placental and decidual tissues. Cytokeratin 7 (KRT7) and HLA-G were used to mark all CTB subtypes and EVTs. Nuclei are stained with DAPI. Representative examples showing localisation of Notch2 in cell column trophoblasts (first panel; dCCT, distal cell column trophoblast; pCCT, proximal cell column trophoblast; 12th week placenta, scale bars represent 100 μm), in interstitial cytotrophoblasts (iCTBs) of the decidua basalis (second panel; 12th week, scale bars represent 50 μm), and in intramural cytotrophoblasts (imCTB), associated with maternal blood vessels (fourth panel; 12th week decidua basalis, digitally zoomed). In all placentae analysed (n = 4, between 6th and 12th week of gestation) expression was weaker in pCCTs and villous cytotrophoblasts (vCTB) compared to non-proliferative, HLA-G + dCCTs. Inserts (i) depict digital magnifications, showing HLA-G and Notch2 in dCCTs. Notch2 was also present in the villous core (VC) as well as in decidual stromal cells (DSC), some of which showed nuclear staining as published . VE-cadherin (CDH5) stained endothelial cells (EC) in spiral arteries (SA) present in maternal decidua (third panel; 12th week decidua basalis, scale bars represent 100 μm). Partial disruption of the maternal EC layer/VE-cadherin staining depicted in (ii) suggested on-going vessel remodelling. imCTBs in (ii) and (iii) showed nuclear Notch2 staining. Selected areas (ii) and (iii) on the right-hand side represent magnified overlays of KRT7 + /Notch2 + imCTBs with nuclear N2ICD.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Expressing, Staining, Disruption

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: Expression of Notch2 and EVT markers in EGFR + and HLA-G + CTBs isolated from first trimester placental tissue. (A) Transcript levels of Notch2 , EGFR , HLA-G , integrin α1 ( ITGA1 ) and integrin α5 ( ITGA5 ) in the two different CTB populations using qRT-PCR. Mean values ± S.D. obtained from five different CTB isolations are shown. For relative quantification (AU, arbitrary units) values of each target gene were arbitrarily set to 1 in EGFR + CTBs. *p < 0.05. Consistent with EVT differentiation HLA-G , ITGA1 and ITGA5 mRNAs were elevated in HLA-G + CTBs compared to EGFR + CTBs, the latter expressing high levels of EGFR . (B) Protein levels of Notch2, EGFR, HLA-G and TCF-4 in the two purified CTB cell types analysed by Western blotting. In agreement with the mRNA expression data HLA-G + CTBs expressed increased levels of Notch2, HLA-G and TCF-4. The latter has been recently established as a marker of EVT . GAPDH was used as loading control.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Quantitative Proteomics, Purification, Western Blot, Marker, Control

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: siRNA-mediated Notch2 knockdown or antibody-mediated inhibition of Notch2-dependent signalling increase motility of primary CTBs and SGHPL-5 cells through fibronectin-coated transwells. (A) Migration in the presence of Notch2 siRNA (siN2) or non-targeting control (ntc). Bars represent mean values ± S.D. obtained from each three CTB isolations/SGHPL-5 cell experiments performed in duplicates. ntc was arbitrarily set to 100%. *p < 0.05. (B) Migration upon addition of Notch2 blocking antibody (N2AB) or IgG control (IgG ctrl). Bars represent mean values ± S.D. obtained from three experiments performed in duplicates. IgG control was arbitrarily set to 100%. *p < 0.05.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Knockdown, Inhibition, Migration, Control, Blocking Assay

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: Antibody-mediated inhibition of Notch2-dependent signalling in first trimester floating villous explant cultures and primary CTBs. Proliferation was analysed by EdU incorporation and counting of the EdU/DAPI ratio. (A) Representative pictures (scale bars represent 50 μm) showing immunofluorescent EdU staining in floating explant cultures treated with Notch2 blocking antibody (N2AB) or IgG control (IgG ctrl). Upper and lower panel show EdU labelling in HAI1 + vCTBs and KRT7 + CCTs. Nuclei are stained with DAPI. EdU + vCTBs and CCTs are indicated by arrows. CCT, cell column trophoblast; VC, villous core; vCTB, villous cytotrophoblasts. (B) Percentage of EdU + villous CTBs and CCTs. Each 14 explants isolated from three different placentae were evaluated in the presence of N2AB or IgG ctrl. Bar graphs represent mean values ± S.D. of 1200–1400 nuclei of vCTBs and 2400–2600 nuclei of CCTs per condition. (C) Percentage of EdU + primary CTBs after treatment with N2AB or IgG ctrl. Bar graphs represent mean values ± S.D. of three experiments performed in duplicates. n.s., not significant.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Inhibition, Staining, Blocking Assay, Control, Isolation

Journal: Journal of Molecular Cell Biology

Article Title: MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth

doi: 10.1093/jmcb/mjy002

Figure Lengend Snippet: Identification of Notch2 as a binding partner of MINAR1. (A) PAE cells expressing empty vector or Myc-tagged MINAR1 were lysed, immunoprecipitated with anti-Myc antibody, resolved in SDS-PAGE. The corresponding band was cut and subjected to proteolytic digestion followed by LC-MS/MS analysis of the tryptic peptides. Shown here is the MS2 higher energy collisional dissociation (HCD) spectrum corresponding to Notch2 peptide ‘MNDGTTPLILAAR’, labeled with assignments for the detected N-terminal b-ion (red) and C-terminal y-ion (blue) fragments. (B) HEK-293 cells expressing FLAG-tagged MINAR1 alone or transfected with Notch2 were lysed and immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-Notch2 antibody. (C) Cell lysates from HUVECs were immunoprecipitated with anti-MINAR1 antibody or control IgG followed by immunoblotting with anti-Notch2 antibody. (D) HUVECs were transduced with two different MINAR1 shRNAs. After 48 h, cells were lysed and subjected to immunoprecipitation using anti-MINAR1 antibody, followed by immunoblotting with anti-Notch2 antibody. (E) PAE cells expressing MINAR1 were subjected to immunofluorescence staining for MINAR1 and Notch2. (F) Whole-cell lysates (WCL) from HEK-293 cells expressing MINAR1 alone or co-expressing MINAR1 with Notch2 were subjected to a filter trap assay as in Figure ​Figure11 and immunoblotted with anti-MINAR1 antibody. WCL of HEK-293 cells expressing MINAR1 alone or co-expressing MINAR1 with Notch2 were subjected to western blot analysis and blotted for MINAR1, Notch2, and the loading control tubulin.

Article Snippet: Notch2 blocking antibody (NOTCH2-B9) was purchased from Addgene ( Falk et al., 2012 ).

Techniques: Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Labeling, Transfection, Western Blot, Control, Transduction, Immunofluorescence, Staining, TRAP Assay

Journal: Journal of Molecular Cell Biology

Article Title: MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth

doi: 10.1093/jmcb/mjy002

Figure Lengend Snippet: Blocking Notch2 antibody inhibits the effect of MINAR1 in tube formation of endothelial cells. PAE cells expressing empty vector or MINAR1 were subjected to a matrigel capillary tube formation assay treated with conditioned medium containing blocking anti-Notch2 antibody or control medium. Pictures were taken after overnight and quantified via Image J/angiogenesis assay.

Article Snippet: Notch2 blocking antibody (NOTCH2-B9) was purchased from Addgene ( Falk et al., 2012 ).

Techniques: Blocking Assay, Expressing, Plasmid Preparation, Capillary Tube Formation Assay, Control, Angiogenesis Assay

Journal: Journal of Molecular Cell Biology

Article Title: MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth

doi: 10.1093/jmcb/mjy002

Figure Lengend Snippet: Identification of Notch2 as a binding partner of MINAR1. (A) PAE cells expressing empty vector or Myc-tagged MINAR1 were lysed, immunoprecipitated with anti-Myc antibody, resolved in SDS-PAGE. The corresponding band was cut and subjected to proteolytic digestion followed by LC-MS/MS analysis of the tryptic peptides. Shown here is the MS2 higher energy collisional dissociation (HCD) spectrum corresponding to Notch2 peptide ‘MNDGTTPLILAAR’, labeled with assignments for the detected N-terminal b-ion (red) and C-terminal y-ion (blue) fragments. (B) HEK-293 cells expressing FLAG-tagged MINAR1 alone or transfected with Notch2 were lysed and immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-Notch2 antibody. (C) Cell lysates from HUVECs were immunoprecipitated with anti-MINAR1 antibody or control IgG followed by immunoblotting with anti-Notch2 antibody. (D) HUVECs were transduced with two different MINAR1 shRNAs. After 48 h, cells were lysed and subjected to immunoprecipitation using anti-MINAR1 antibody, followed by immunoblotting with anti-Notch2 antibody. (E) PAE cells expressing MINAR1 were subjected to immunofluorescence staining for MINAR1 and Notch2. (F) Whole-cell lysates (WCL) from HEK-293 cells expressing MINAR1 alone or co-expressing MINAR1 with Notch2 were subjected to a filter trap assay as in Figure ​Figure11 and immunoblotted with anti-MINAR1 antibody. WCL of HEK-293 cells expressing MINAR1 alone or co-expressing MINAR1 with Notch2 were subjected to western blot analysis and blotted for MINAR1, Notch2, and the loading control tubulin.

Article Snippet: Notch2 blocking antibody (NOTCH2-B9) was purchased from Addgene ( Falk et al., 2012 ).

Techniques: Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Labeling, Transfection, Western Blot, Transduction, Immunofluorescence, Staining, TRAP Assay

Journal: Journal of Molecular Cell Biology

Article Title: MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth

doi: 10.1093/jmcb/mjy002

Figure Lengend Snippet: Blocking Notch2 antibody inhibits the effect of MINAR1 in tube formation of endothelial cells. PAE cells expressing empty vector or MINAR1 were subjected to a matrigel capillary tube formation assay treated with conditioned medium containing blocking anti-Notch2 antibody or control medium. Pictures were taken after overnight and quantified via Image J/angiogenesis assay.

Article Snippet: Notch2 blocking antibody (NOTCH2-B9) was purchased from Addgene ( Falk et al., 2012 ).

Techniques: Blocking Assay, Expressing, Plasmid Preparation, Capillary Tube Formation Assay, Angiogenesis Assay

Journal: PLoS ONE

Article Title: Notch2 Controls Prolactin and Insulin-Like Growth Factor Binding Protein-1 Expression in Decidualizing Human Stromal Cells of Early Pregnancy

doi: 10.1371/journal.pone.0112723

Figure Lengend Snippet: Serial sectioning of paraffin-embedded tissues and immunofluorescence were performed as described in . Representative examples (8 th week of pregnancy) of 5 different deciduae analyzed are depicted. DSC, decidual stromal cells; GEC, glandular epithelial cell; LC, leukocytes; Scale bars represent 100 µm. (A) Localization of Notch family members in human deidual stromal cells. Double staining with antibodies recognizing vimentin (Vim, green), Notch2 (green) and CD45 (red) mark decidual stromal cells and leukocytes, respectively. The respective counterstaining with DAPI is depicted on the right hand side. Double stainings with the appropriate isotype-specific monoclonal (mAb, green, insert picture) and polyclonal (pAb, green, insert picture) controls with vimentin (Vim, red) are shown. (B) Glandular expression of Notch receptors and ligands. Double staining with antibodies recognizing vimentin (Vim, green) or cytokeratin 7 (KRT7, red) was used to depict DSC and GEC, respectively. Co-staining of Notch receptor or ligand (both green) with nuclear staining (DAPI, blue) is shown.

Article Snippet: To specifically inhibit the Notch2 receptor, seeded cells were treated with 0.4 μg/ml human IgG isotype control (Novus Biologicals) or 0.4 μg/ml therapeutic human Notch2 IgG 1 blocking antibody, which binds and stabilizes the auto-inhibited, negative regulatory region (NRR) of the receptor, preventing conformational changes and therefore ADAM-mediated cleavage .

Techniques: Immunofluorescence, Double Staining, Expressing, Staining

Journal: PLoS ONE

Article Title: Notch2 Controls Prolactin and Insulin-Like Growth Factor Binding Protein-1 Expression in Decidualizing Human Stromal Cells of Early Pregnancy

doi: 10.1371/journal.pone.0112723

Figure Lengend Snippet: Cells were stimulated with cAMP and/or E2P4 for 3 and 6 days. Total protein lysates were analyzed by western blotting and quantitated using densitometry as described in . A representative example out of 3 different experiments is shown. n.c., non-stimulated controls; (A) Western blot showing specific signals for Notch2, Jagged1, DLL1 and DLL4 (marked by arrows). GAPDH was used as a loading control, * indicates unspecific band. (B) Densitometrical quantification of western blot signals normalized to GAPDH. Bars (n = 3) represent mean values ± S.D. For relative quantification of protein expression, values of n.c. at day 3 were arbitrarily set to 100%. * indicates p≤0.05 compared to n.c. of day 3; n.s., not significant.

Article Snippet: To specifically inhibit the Notch2 receptor, seeded cells were treated with 0.4 μg/ml human IgG isotype control (Novus Biologicals) or 0.4 μg/ml therapeutic human Notch2 IgG 1 blocking antibody, which binds and stabilizes the auto-inhibited, negative regulatory region (NRR) of the receptor, preventing conformational changes and therefore ADAM-mediated cleavage .

Techniques: Western Blot, Control, Quantitative Proteomics, Expressing

Journal: PLoS ONE

Article Title: Notch2 Controls Prolactin and Insulin-Like Growth Factor Binding Protein-1 Expression in Decidualizing Human Stromal Cells of Early Pregnancy

doi: 10.1371/journal.pone.0112723

Figure Lengend Snippet: Cells were cultured in the absence or presence of Notch2 blocking antibodies (Notch2 ab) (A) or si-RNAs targeting Notch2 (si-Notch2) (B). (A) Luciferase activity of the canonical Notch reporter in the presence of Notch2 ab or IgG controls. Reporter expression was normalized to constitutive β-Gal activity. Mean values ± S.D. of 3 experiments performed in duplicates are shown. IgG ctrl, IgG control; * indicates p≤0.05 compared to IgG-treated n.c. Significant changes (*, p≤0.05) between DAPT-treated (open bars) and untreated (black bars) cells are indicated by brackets. (B) Western blot showing Notch2 protein expression in siRNA-treated HDSC. Specific Notch2 signals are marked by an arrow (110 kDa). GAPDH was used as a loading control. Representative examples of 3 independent experiments are depicted. Lower panel shows the densitometrical quantification calculated from 3 independent experiments. si-ntc, si-non targeting control. * indicates p≤0.05 compared to si-ntc of the same day.

Article Snippet: To specifically inhibit the Notch2 receptor, seeded cells were treated with 0.4 μg/ml human IgG isotype control (Novus Biologicals) or 0.4 μg/ml therapeutic human Notch2 IgG 1 blocking antibody, which binds and stabilizes the auto-inhibited, negative regulatory region (NRR) of the receptor, preventing conformational changes and therefore ADAM-mediated cleavage .

Techniques: Cell Culture, Blocking Assay, Luciferase, Activity Assay, Expressing, Control, Western Blot

Journal: PLoS ONE

Article Title: Notch2 Controls Prolactin and Insulin-Like Growth Factor Binding Protein-1 Expression in Decidualizing Human Stromal Cells of Early Pregnancy

doi: 10.1371/journal.pone.0112723

Figure Lengend Snippet: HDSC were differentiated with cAMP/E2P4 for 6 days in the absence or presence of Notch2-blocking antibodies or siRNAs as mentioned in . Quantitative real-time PCR analyses showing mRNA expression of the Notch target gene HES1 and the markers of decidualization, IGFBP1 and PRL, upon addition of Notch2 blocking antibodies (A) or Notch2 siRNAs (B). For relative quantification non-stimulated controls (n.c.) were arbitrarily set to 1. Bars indicate mean values ± S.D. of 3 different experiments performed in duplicates. * indicates p≤0.05 compared to n.c.; Significant changes (*, p≤0.05) between Notch2 ab or si-Notch2-treated cells (open bars) and untreated (black bars) cells are indicated by brackets. IgG ctrl, IgG control; si-ntc, si-non targeting control.

Article Snippet: To specifically inhibit the Notch2 receptor, seeded cells were treated with 0.4 μg/ml human IgG isotype control (Novus Biologicals) or 0.4 μg/ml therapeutic human Notch2 IgG 1 blocking antibody, which binds and stabilizes the auto-inhibited, negative regulatory region (NRR) of the receptor, preventing conformational changes and therefore ADAM-mediated cleavage .

Techniques: Blocking Assay, Real-time Polymerase Chain Reaction, Expressing, Quantitative Proteomics, Control

Journal: PLoS ONE

Article Title: Notch2 Controls Prolactin and Insulin-Like Growth Factor Binding Protein-1 Expression in Decidualizing Human Stromal Cells of Early Pregnancy

doi: 10.1371/journal.pone.0112723

Figure Lengend Snippet: Cells were incubated with Notch2 blocking antibodies (A) or Notch2 siRNAs (B) and differentiated for 6 days in the presence of cAMP/E2P4. Supernatants were collected and prolactin (PRL) concentrations were measured by ELISA. Normalization to cellular protein concentrations was performed as mentioned in . Bars represent mean values ± S.D. of each 3 different experiments performed in duplicates. * indicates p≤0.05 compared to non-stimulated control (n.c.); Significant changes (*, p≤0.05) between Notch2 ab or si-Notch2-treated cells (open bars) and untreated (black bars) cells are indicated by brackets. IgG ctrl, IgG control; si-ntc, si-non targeting control.

Article Snippet: To specifically inhibit the Notch2 receptor, seeded cells were treated with 0.4 μg/ml human IgG isotype control (Novus Biologicals) or 0.4 μg/ml therapeutic human Notch2 IgG 1 blocking antibody, which binds and stabilizes the auto-inhibited, negative regulatory region (NRR) of the receptor, preventing conformational changes and therefore ADAM-mediated cleavage .

Techniques: Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Control