goat anti-notch4  (Santa Cruz Biotechnology)

Journal: Blood

Article Title: Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7

doi: 10.1182/blood-2010-03-274860

Figure Lengend Snippet: EGFL7 functions as an antagonist of Notch in HUVECs. (A) Human EGFL7 expression in lentivirally generated HUVEC lines, as measured by quantitative RT-PCR. Empty lentiviruses pCCL GFP and pLKO served as controls for EGFL7 overexpression and knockdown, respectively. Data are represented as fold induction relative to the lentiviral controls. (B) Proliferation assay of HUVEC lines grown in complete medium for 4 days. (C) Capillary sprouting assay. Control (GFP and pLKO), EGFL7 overexpressing (EGFL7), and knockdown (pLKO 61) HUVEC lines were coated on a cytodex bead, beads embedded in fibrin gels, and visualized on day 7. (D) HUVEC monolayer wounding assay at 0 and 24 hours. Dotted lines highlight the edges of the monolayer. (E) Quantitation of HUVEC migration in monolayer wounding assay, represented as percentage of area filled at 8 hours. (F) Transactivation of Notch/CSL-luciferase reporter in EGFL7 overexpressing and knockdown HUVEC lines. Data represented as relative luciferase units (RLU). (G) Notch4-dependent HUVEC morphogenesis assay. Control HUVECs (pCCL-GFP or pLKO) or EGFL7 overexpressing (EGFL7) or knockdown (pLKO61) cells were grown as monolayer on a fibrin gel. GFP- or Notch4/GFP-expressing HUVECs were overlaid on top of the monolayer. At day 7, cocultures were visualized and the number of GFP+ cells undergoing morphogenesis, as seen by the extending of processes into the surrounding matrix per field, was determined. Experiments were performed in triplicate, and error bars represent SD.

Article Snippet: Western blotting Samples were boiled at 100°C for 5 minutes, resolved, blotted on polyvinylidene difluoride membranes, and probed using either goat anti–human EGFL7 (R-12) antibody (Santa Cruz Biotechnology Sc34416, 1:1000), hamster anti–mouse Notch1 (Millipore MAB5414, 1:1000), goat anti–mouse Notch4 (E11; Santa Cruz Biotechnology Sc32634, 1:1000), or mouse anti–β-tubulin (Sigma-Aldrich T4026, 1:1000).

Techniques: Expressing, Generated, Quantitative RT-PCR, Over Expression, Proliferation Assay, Quantitation Assay, Migration, Luciferase

Journal: Blood

Article Title: Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7

doi: 10.1182/blood-2010-03-274860

Figure Lengend Snippet: EGFL7 interacts with Notch receptors and regulates Notch target gene expression in vivo. (A) Alignment of the DSL domain of Jagged1, Serrate, Delta, and Lag-2 with the putative DSL domain in EGFL7. Red letters represent the consensus sequence. (B) Yeast-2-hybrid assay (left panel): EGFL7 interacts with NOTCH4 and DLL4. Full-length EGFL7, DLL4, or the extracellular domain of NOTCH4 were fused to either the DNA-binding domain or the transcriptional activation domain of GAL4, and protein-protein interactions were monitored by the ability of the transformed yeast to grow on defined medium, and expression of α-galactosidase. Yeast-3-hybrid assay (right panels): EGFL7 abolishes NOTCH4-DLL4 interaction. The Egfl7 ORF was cloned downstream of a methionine repressible promoter (Met25) and transformed into a yeast strain expressing Notch4-GAL activating domain and DLL4-GAL4 DNA-binding domain fusions. Expression of α-galactosidase was then assayed on X-gal selection plates with or without methionine. (Ci-ii) Coimmunoprecipitation assays with protein extracts prepared from HEK293 cells transfected with plasmids encoding MYC/His-tagged-Egfl7 and Notch4 ECD or Notch1 ECD. (i) A NOTCH4 antibody was used to immunoprecipitate NOTCH4 ECD, and protein complexes were probed for NOTCH4 and EGFL7 by Western blot. (ii) A MYC antibody was used to immunoprecipitate EGFL7, and protein complexes were probed for NOTCH1 and EGFL7 by Western blot. (iii) Coimmunoprecipitation assay with protein extracts prepared from HUVECs infected with an adenovirus encoding MYC-tagged-Egfl7. A MYC antibody was used to immunoprecipitate EGFL7, and protein complexes were probed for NOTCH1 and EGFL7 by Western blot. (iv) Coimmunoprecipitation assays using protein lysates prepared from E12.5 embryos. An antibody against NOTCH4 was used to immunoprecipitate NOTCH4, and protein complexes were probed for NOTCH4 and EGFL7 by Western blot. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Notch target gene expression in wild-type (□, n = 4) and Tie2-Egfl7 transgenic (■, n = 6) retinas. Gene expression was measured by quantitative RT-PCR and normalized to endothelial cell number using CD31 expression. (E) Notch target gene expression in wild-type (white bars, n = 6) and Tie2-Egfl7 transgenic embryos (black bars, n = 4). Data are represented as fold change compared with wild-type. *P < .05.

Article Snippet: Western blotting Samples were boiled at 100°C for 5 minutes, resolved, blotted on polyvinylidene difluoride membranes, and probed using either goat anti–human EGFL7 (R-12) antibody (Santa Cruz Biotechnology Sc34416, 1:1000), hamster anti–mouse Notch1 (Millipore MAB5414, 1:1000), goat anti–mouse Notch4 (E11; Santa Cruz Biotechnology Sc32634, 1:1000), or mouse anti–β-tubulin (Sigma-Aldrich T4026, 1:1000).

Techniques: Expressing, In Vivo, Sequencing, Y2H Assay, Binding Assay, Activation Assay, Transformation Assay, Hybrid Assay, Clone Assay, Selection, Transfection, Western Blot, Co-Immunoprecipitation Assay, Infection, Transgenic Assay, Quantitative RT-PCR