Journal: Brain

Article Title: Notch-1 signalling is activated in brain arteriovenous malformations in humans

doi: 10.1093/brain/awp246

Figure Lengend Snippet: Immunohistochemical detection of Notch-1 ligands and Hes-1 in human brain AVMs. (A) Dll4, Jagged-1 and Hes-1 expression (black) was increased in human brain AVMs (left panels, low-magnification; middle panels, high-magnification) compared with normal human middle cerebral artery (right panels). A few non-vascular cells in normal control brain also expressed Hes-1 (arrow). (B) Notch1, Jagged-1, Dll4, NICD and Hes-1 were expressed or activated in vascular cells of E17 mouse brain. (C) Immunostaining was performed on H9 human embryonic stem cells using (left to right) anti-Jagged-1 (green) and anti-NICD (red); anti-Notch-1 (green); anti-Dll4 (green) and anti-Hes1 (green). DAPI (blue) was used to counterstain nuclei.

Article Snippet: Recombinant human Notch-1/Fc chimera, recombinant human Jagged-1/Fc chimera, recombinant human Dll4 and Hes-1 protein (all from R&D System) were used as positive controls.

Techniques: Immunohistochemical staining, Expressing, Immunostaining

Journal: Brain

Article Title: Notch-1 signalling is activated in brain arteriovenous malformations in humans

doi: 10.1093/brain/awp246

Figure Lengend Snippet: Western blot analysis and cell-type localization of Notch-1 ligands in human brain AVMs. (A) Protein was isolated from the sources listed in the legend to Figure 1, and from normal mouse cerebral cortex (M-CTX). Western blotting was performed using antibodies against the Notch-1 ligands Dll4 (top panel) and Jagged-1 (middle panel), with anti-actin to control for differences in protein loading (bottom panel). (B) Recombinant human Notch-1, Jagged-1, Dll4 and Hes1 were loaded on gels and probed with the indicated antibodies, showing that the antibodies used recognize authentic antigens. Each pair of gel lanes was loaded with 100 ng (lane 1) or 500 ng (lane 2) of total protein. (C) Double-label immunostaining of human brain AVM sections shows that αSMC-expressing vascular smooth muscle cells also express Jagged-1 (top panel) and Dll4 (bottom panel). DAPI (blue) was used to counterstain nuclei. (D) Triple-label immunostaining of human brain AVM shows co-expression of Jagged-1 (red) and Hes1 (green) in CD31-immunopositive (purple) endothelial cells. DAPI (blue) was used to counterstain nuclei.

Article Snippet: Recombinant human Notch-1/Fc chimera, recombinant human Jagged-1/Fc chimera, recombinant human Dll4 and Hes-1 protein (all from R&D System) were used as positive controls.

Techniques: Western Blot, Isolation, Recombinant, Immunostaining, Expressing

Journal: Endocrinology

Article Title: Activation of Notch Signaling by Oocytes and Jag1 in Mouse Ovarian Granulosa Cells

doi: 10.1210/en.2019-00564

Figure Lengend Snippet: Effects of recombinant Jag1 protein on Notch target gene expression in granulosa cells. Primary cultured granulosa cells were plated onto immobilized recombinant JAG1 (rJ1) overnight as described in the “Methods” section and treated with vehicle or the Notch inhibitor DAPT for 24 h before isolation of RNA. Expression of the Notch target/effector genes (A) Hes1, (B) Hey2, and (C) Heyl was analyzed. n = 3 for each condition, with each biological replicate indicated as a point on the scatter plots. The bars designate mean ± SEM. Means not sharing the same letter are significantly different (at P < 0.05).

Article Snippet: Primary granulosa cell culture and recombinant JAG1 ligand To make recombinant JAG1–coated substrate, tissue culture plates were incubated with a solution of 5 µg/mL recombinant rat Jagged1-Fc chimeric protein (R&D Systems, Minneapolis, MN) and 1 µg/cm 2 fibronectin (Sigma-Aldrich, St. Louis, MO) overnight at 4°C with shaking to allow for protein adsorption.

Techniques: Recombinant, Expressing, Cell Culture, Isolation, RNA Expression

Journal: Endocrinology

Article Title: Activation of Notch Signaling by Oocytes and Jag1 in Mouse Ovarian Granulosa Cells

doi: 10.1210/en.2019-00564

Figure Lengend Snippet: TNR (EGFP) expression in the ovaries of oocyte-specific Jag1 knockout mice. (A) Histology of WT and knockout (J1KO) ovaries at PND10, showing the presence of oocytes in the knockout ovaries. Scale bars, 100 µm. (B) Gene expression analysis indicating levels of Jag1 and Egfp mRNAs in normal littermates (NLM) (open bars) or cJ1KO/TNR (filled bars) ovaries at PND10. n = 4, with each animal shown as a point in the scatter plots. (C) Gene expression analysis indicating levels of Hes1, Hey2, and Heyl mRNAs in NLM (open bars) or cJ1KO/TNR (filled bars) ovaries at PND10. n = 5, with each animal an individual point in the scatter plots. The bars designate mean ± SEM. *P < 0.05.

Article Snippet: Primary granulosa cell culture and recombinant JAG1 ligand To make recombinant JAG1–coated substrate, tissue culture plates were incubated with a solution of 5 µg/mL recombinant rat Jagged1-Fc chimeric protein (R&D Systems, Minneapolis, MN) and 1 µg/cm 2 fibronectin (Sigma-Aldrich, St. Louis, MO) overnight at 4°C with shaking to allow for protein adsorption.

Techniques: Expressing, Knock-Out

Journal: Endocrinology

Article Title: Activation of Notch Signaling by Oocytes and Jag1 in Mouse Ovarian Granulosa Cells

doi: 10.1210/en.2019-00564

Figure Lengend Snippet: Effects of recombinant JAG1 protein on Notch ligand and receptor gene expression in granulosa cells. Primary cultured granulosa cells were plated onto fibronectin (control) or immobilized recombinant Jag1 as described in the “Methods” section. (A) Expression of the Notch ligands Jag1 and Jag2 and the Notch receptors Notch2 and Notch3 was analyzed. (B) Expression of the ligand Jag1 was analyzed with or without a 24-h treatment with the Notch inhibitor DAPT. n = 3 for each condition, with individual experiments shown as points on the scatter plots. The bars designate mean ± SEM. In (A), *P < 0.05. In (B), means not sharing the same letter are significantly different (at P < 0.05).

Article Snippet: Primary granulosa cell culture and recombinant JAG1 ligand To make recombinant JAG1–coated substrate, tissue culture plates were incubated with a solution of 5 µg/mL recombinant rat Jagged1-Fc chimeric protein (R&D Systems, Minneapolis, MN) and 1 µg/cm 2 fibronectin (Sigma-Aldrich, St. Louis, MO) overnight at 4°C with shaking to allow for protein adsorption.

Techniques: Recombinant, Expressing, Cell Culture