notch2 blocking antibody notch2-b9  (Addgene inc)

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