Journal: Development (Cambridge, England)

Article Title: ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning

doi: 10.1242/dev.200528

Figure Lengend Snippet: DLL4 expression and tip cell positioning are associated with ECFAK deficiency, FAK-kinase activity and phosphorylation at FAK-Y397 . Imaris 3D visualisation of DLL4/IB4 double-stained P6 retinal angiogenic fronts and DLL4 quantification in vivo in (A) ECFAK WT and ECFAK KO and (B) ECFAK KIWT , ECFAK KD , ECFAK Y397F and ECFAK Y861F mice. n =4-6 mice in A and 3-6 mice in B. Data were analysed with a Student's t -test in A and one-way ANOVA in B. (C) Confocal images and quantification of in vitro sprouting competition assays of differentially labelled (green or red dye) FAK WT and FAK KO or (D) FAK KIWT , FAK KD , FAK Y397F and FAK Y861F mutant EC lines. Asterisks indicate ECs at the tip position. n =10-20 spheroids per condition from two independent experiments. (E) VEGF-induced migration competition assays in vitro between FAK KIWT (green) and FAK KIWT , FAK KD , FAK 397F or FAK 861F (red) primary mouse lung ECs. Forward Migration Index (FMI) quantifications are shown. n =3 independent experiments analysing 31-60 cells per condition in each. Data are mean± s.e.m. analysed using a two-way ANOVA; * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001; NS, not significant in A-E. Scale bars: 50 µm.

Article Snippet: Real-time PCR was performed in a StepOne Plus thermocycler (4376357, Applied Biosystems) using TaqMan Master mix (4369514, Applied Biosystems) and primers custom-made that were specific to mouse Fak or chicken Fak , Dll4 (Mm00444619_m1), Nrarp (Mm00482529_s1), Hey1 (Mm00468865_m1), Hey2 (Mm00469280_m1), Erg (Mm01214244_m1), Pecam1/CD31 (Mm01246167_m1) and Gapdh (4352339E) (all from Applied Biosystems).

Techniques: Expressing, Activity Assay, Phospho-proteomics, Staining, In Vivo, In Vitro, Mutagenesis, Migration

Journal: Development (Cambridge, England)

Article Title: ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning

doi: 10.1242/dev.200528

Figure Lengend Snippet: ERG re-expression in ECFAK KO restores DLL4 levels and vascular outgrowth in vivo and tip cell positioning and directed chemotactic migration in vitro . (A) IB4/ERG/DLL4 triple-stained retinal fronts and quantification in Ad- lacZ or Ad-ERG P6 ECFAK WT and ECFAK KO pups. (B) ERG and (C) DLL4 in Ad- lacZ or Ad-ERG P6 ECFAK WT and ECFAK KO pups transduced in vivo . (D) Vascular outgrowth analysis. n =3 mice per genotype. (E) Representative images and (F) quantification of a sprouting competition assay of FAK WT and FAK KO ECs nucleofected with control (pControl) or ERG (pERG) plasmids in vitro. n =10-20 spheroids per condition. (G) Dunn chamber chemotaxis, pControl- or pERG- FAK WT and FAK KO ECs. (H) Speed and (I) FMI analysis . n =3 independent experiments analysing 22-44 cells per condition. (J) Schematic of how ERG regulation by ECFAK affects blood vessel development and patterning. Data are mean±s.e.m., analysed using a two-way ANOVA, with ** P <0.01; *** P <0.001; **** P <0.0001; # 0.0642; NS, not significant. Scale bars: 50 µm.

Article Snippet: Real-time PCR was performed in a StepOne Plus thermocycler (4376357, Applied Biosystems) using TaqMan Master mix (4369514, Applied Biosystems) and primers custom-made that were specific to mouse Fak or chicken Fak , Dll4 (Mm00444619_m1), Nrarp (Mm00482529_s1), Hey1 (Mm00468865_m1), Hey2 (Mm00469280_m1), Erg (Mm01214244_m1), Pecam1/CD31 (Mm01246167_m1) and Gapdh (4352339E) (all from Applied Biosystems).

Techniques: Expressing, In Vivo, Migration, In Vitro, Staining, Competitive Binding Assay, Control, Chemotaxis 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: 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: Quantitative real-time PCR was performed on the ABI 7500 Sequence Detection System (Applied Biosystems, Carlsbad, CA) using Taq Man Gene Expression Assays (TaqMan Universal PCR Master Mix, 20x Taq Man Gene Expression Assay Mix for Notch1 (Hs01062014_m1), Notch2 (Hs01050702_m1), Notch3 (Hs01128541_m1), Notch4 (Hs00965889_m1), Jagged1 (Hs01070032_m1), Jagged2 (Hs00171432_m1), DLL1 (Hs00194509_m1), DLL3 (Hs01085096_m1), DLL4 (Hs00184092_m1), HES1 (Hs00172878_m1), PRL (Hs00168730_m1), IGFBP1 (Hs00426285_m1) and TATA-box binding protein (TBP; TaqMan endogenous control)) according to the manufacturer's instructions.

Techniques: Western Blot, Control, Quantitative Proteomics, Expressing

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: NIH3T3 cells expressing mNotch1 (a–c) or mNotch2 (d–f) were co-cultured with cells expressing Dll1 (a, d), Dll4 (b, e), or Jag1 (c, f) in the presence of peracetylated fucose analogs (compounds 9, 10, 11 ). Cells transfected with empty vector (EV) were used as a negative control and cells grown in DMSO were a positive control for Notch signaling. All experiments were performed three independent times, and data represents nine biological replicates (n=9). All plots represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, Tukey post-hoc test, adjusted p values.

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Expressing, Cell Culture, Transfection, Plasmid Preparation, Negative Control, Positive Control

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: Notch ligand binding experiments were performed to measure the effect of fucose analogs on the ability of mNotch1 (a–c) or mNotch2 (d–f) transfectants to bind soluble Dll1-Fc (a, d), Dll4-Fc (b, e), or Jag1-Fc (c, f) using cell-based flow cytometry assays. EV transfected cells were used as a negative control and cells cultured in the presence of DMSO served as a positive control. Six independent experiments were performed for all samples (n=6). All plots represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, Tukey post-hoc test, adjusted p values.

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Ligand Binding Assay, Flow Cytometry, Transfection, Negative Control, Cell Culture, Positive Control

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: (a) Notch1 activation assays using Dll1-Fc (left) or Dll4-Fc (right) as activating ligands coated on plates showing the effect of mutations at EGF8, 12 or both on Dll1 and Dll4-induced Notch1 signaling. Relative Luciferase Unit (RLU) normalized to wild type (WT) Notch1. (b) Plate coating Notch1 activation assays using Dll1-Fc as activating ligand in the presence of DMSO (control) or 100 nM of the indicated compound. RLU for all constructs (WT and mutants) in DMSO was normalized to 1. (c) Plate coating assays for Dll4-induced signaling using 50 μM fucose analogs. The EGF8/12 double mutant was also tested. RLU for all constructs was normalized to the DMSO control. Box and whisker plots represent six biological replicates (n=6). *p<0.05, **p<0.01, ***p<0.001, Tukey post-hoc test, adjusted p values. (d) Model figure for fucose analog mediated inhibition of Notch1 activation. Elimination of the O -fucose site on EGF8, but not EGF12, partially rescues fucose analog inhibition of Notch1 activation by Dll1 suggesting that binding sites corresponding to O -fucose on EGF12 are tolerant of the additional C6 carbon in compounds 10 and 11 . In contrast, the binding site on Dll1 is not tolerant of the additional carbon at EGF8, creating a steric clash, resulting in the observed decreased activation of Notch1. Additional sites contain O -fucose consensus sequences and are modified with O -fucose (red ovals), but contribute little to Notch1-ligand interactions compared to EGF8 and EGF12 .

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Activation Assay, Luciferase, Construct, Mutagenesis, Whisker Assay, Inhibition, Binding Assay, Modification

Journal: Nature chemical biology

Article Title: Inhibition of Delta-induced Notch signaling using fucose analogs

doi: 10.1038/nchembio.2520

Figure Lengend Snippet: Representative flow cytometric profiles of cells produced from bone marrow LSK cells co-cultured with OP9-GFP, OP9-Dll1 or OP9-Dll4 stromal cells in the presence of DMSO, compound 9 or compound 10 for 8 days. (a) Production of CD25 + T-cell progenitors was evaluated by the expression of CD44 and CD25. (c) Percentage of CD25 + T-cells from mice with a profile typical of panel a (n=3). Mean ± SEM, each symbol represents average data from duplicate wells of LSK cells from one mouse. Data shown are representative of three experiments performed in duplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, unpaired two-tailed Student’s t-test.

Article Snippet: Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (each at 0.5 μg/mL) were pre-clustered with fluorescent secondary antibody PE-goat anti-mouse IgG (1:100; Invitrogen, P-852) or PE-anti-human IgG (1:100; Jackson Immuno Research, 109155-098) for 30 min at 4 °C.

Techniques: Produced, Cell Culture, Expressing, Two Tailed Test

Journal: Chinese Herbal Medicines

Article Title: Qishen Granule protects against myocardial ischemia by promoting angiogenesis through BMP2-Dll4-Notch1 pathway

doi: 10.1016/j.chmed.2023.12.007

Figure Lengend Snippet: Effects of QSG on angiogenesis of BMP2-Dll4-Notch1 pathway in myocardial ischemic rats. (A−B) Representative immunoblot images and quantitative analysis of BMP2, Dll4 and Notch1 in different groups. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. (C) Representative immunofluorescent images were shown. CD31 was used to identify endothelial cells, DAPI to visualize cell nuclei. Scale bar, 100 μm (× 20). Data were presented as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs model group.

Article Snippet: Marker (5 μL) and protein sample (10 μL) were added into the sample hole of the prepared concentrated glue, which was separated at 110 V for 90 min, transferred at 300 mA for 90 min, and blocked with 5% milk for 2 h. The film was incubated in anti-BMP2 (YT5651; Immunoway), anti-Dll4 (bs-5909R; Bioss) and anti-Notch1 (10062–2-AP; ProteinTech) at 4 °C overnight, and anti-rabbit IgG H&L (ab16284; Abcam) was incubated at room temperature for 1 h. The film was placed in a gel imager, the luminescent liquid was uniformly dropped on the film, and the image was obtained and saved.

Techniques: Western Blot, Control

Journal: Chinese Herbal Medicines

Article Title: Qishen Granule protects against myocardial ischemia by promoting angiogenesis through BMP2-Dll4-Notch1 pathway

doi: 10.1016/j.chmed.2023.12.007

Figure Lengend Snippet: Effects of QSG on protein expressions in BMP2-Dll4-Notch1 pathway in OGD-induced HUVECs model. (A) Expression of BMP2 in HUVECs detected by Western blots. (B) Release of BMP2 in supernatant of HUVECs detected by ELISA. (C−D) Representative immunoblots and corresponding quantification of Dll4 and Notch1 protein levels in HUVECs. Data were presented as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 vs model group.

Article Snippet: Marker (5 μL) and protein sample (10 μL) were added into the sample hole of the prepared concentrated glue, which was separated at 110 V for 90 min, transferred at 300 mA for 90 min, and blocked with 5% milk for 2 h. The film was incubated in anti-BMP2 (YT5651; Immunoway), anti-Dll4 (bs-5909R; Bioss) and anti-Notch1 (10062–2-AP; ProteinTech) at 4 °C overnight, and anti-rabbit IgG H&L (ab16284; Abcam) was incubated at room temperature for 1 h. The film was placed in a gel imager, the luminescent liquid was uniformly dropped on the film, and the image was obtained and saved.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A perivascular niche supports endometrial epithelial regeneration

doi: 10.1101/2024.03.07.583958

Figure Lengend Snippet: (A) Immunofluorescence images of P60 CBF :H2B-Venus uterus. Arrow indicates Venus-expressing cells adjacent to the vasculature that express ERα. (B) Scheme of ovariectomized mouse model and treatment. (C) Immunofluorescence images of CBF :H2B-Venus uteri post ovariectomy after various hormonal treatments. (D-F) qRT-PCR analyses showing mRNA levels of Notch target genes (D), Notch receptors (E), and Notch ligands (F). (G) Immunofluorescence images of P60 CBF :H2B-Venus uteri. White arrows point to perivascular Venus-expressing cells that express Notch3, and yellow arrows point to Venus-expressing cells adjacent to DLL4+ vasculature. Scale: 100 μm. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: For DLL4 culture experiments, an 8-well chamber slide (Millipore #PEZGS0816, Burlington, MA) was freshly coated with 2 µg DLL4 (Sino Biological #50640-M08H, Beijing, China) at 37°C for 4 hours.

Techniques: Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A perivascular niche supports endometrial epithelial regeneration

doi: 10.1101/2024.03.07.583958

Figure Lengend Snippet: (A-E) Violin plots from previously published uterine scRNA-seq data showing the distribution and expression levels of Notch2 (A), Notch3 (B), and Dll4 (C) in different cell types in D4 mouse uterus , and Notch3 (D) and Dll4 (E) in different cell types in D8 mouse uterus .

Article Snippet: For DLL4 culture experiments, an 8-well chamber slide (Millipore #PEZGS0816, Burlington, MA) was freshly coated with 2 µg DLL4 (Sino Biological #50640-M08H, Beijing, China) at 37°C for 4 hours.

Techniques: Expressing

Journal: bioRxiv

Article Title: Endothelial Semaphorin 3fb regulates Vegf pathway-mediated angiogenic sprouting

doi: 10.1101/2021.08.03.454978

Figure Lengend Snippet: Sema3fb is expressed by endothelial cells during angiogenic sprout formation and acts through an autocrine mechanism to suppress Vegfr2 expression and maintain endothelial cell dynamics via controlling Dll4 expression and Notch signaling in addition to modulation of pERK. Loss of sema3fb increases Vegfr2, pERK, Dll4, and sFlt1 expression. This results in aberrant cellular morphology with wider sprouts, persistent filopodia, and larger nuclei. The changes in nuclear size and disrupted sprouting suggest a possible role for Sema3b to limit Vegf-mediated induction of downstream pERK signaling.

Article Snippet: Zebrafish specific Taqman assays (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used: vegfab (Cat# 4448892, Clone ID: Dr03072613_m1), kdrl(vegfr2) (4448892, Dr03432904_m1), dll4 (4448892, Dr03428646_m1), notch2 (4448892, Dr03436779_m1), jag1a (4448892, Dr03093490_m1), sflt1 (4331448, ADP47YD4) and normalized to β-actin (4448489, Dr03432610_m1).

Techniques: Expressing

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 1 Immunization with plasmid-encoded DLL4 results in an immunological response in BALB/c mice. (a) Expression of human DLL4 was verified by transient transfection of HeLa cells with empty vector or the vector encoding DLL4 (pDLL4) and analyzed by western blot. Recombinant human DLL4 was used as a positive control (rhDLL4). (b) Female BALB/c mice were immunized either intramuscularly (IM) or intradermally (ID) according to the depicted protocols. All intradermal injections were given in combination with electroporation (EP). One week after the final immunization animals were challenged by orthotopic injection of D2F2/E2 or TUBO cells in the mammary fat pad. (c) To investigate if the vaccination gave rise to an immunological response, serum was taken 10 days after the final immunization (protocol 1) and analyzed by human DLL4 enzyme-linked immunosorbent assay (ELISA). The figure depicts a representative analysis for one out of four mice analyzed.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Plasmid Preparation, Expressing, Transfection, Western Blot, Recombinant, Positive Control, Electroporation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 2 DNA vaccination against DLL4 results in suppression of tumor growth in BALB/c mice. (a) Expression of DLL4 in D2F2/ E2 tumor endothelial cells was assessed by immunofluorescent staining for the endothelial markers, CD31 (red) and DLL4 (green). Cell nuclei were counterstained by DAPI (blue). Growth of orthotopically implanted D2F2/E2 (b and c) or TUBO (d) mammary carcinomas in BALB/c mice following immunization with empty vector or with DLL4 plasmid DNA according to protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP; n ¼ 8 for all groups). The data shown in (b and d) are representative of three independent experiments. *Po0.05, **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Expressing, Staining, Plasmid Preparation

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 3 Targeting DLL4 by vaccination results in excessive formation of nonfunctional blood vessels. (a) Visual inspection of excised D2F2/E2 tumors revealed overt thrombosis in tumors from DLL4-vaccinated mice. (b) Blood vessel density of D2F2/E2 tumors visualized by immunostaining for the endothelial cell marker CD31 (red; n ¼ 5 for each group). (c) Perfused blood vessels of D2F2/E2 tumors as determined by vascular perfusion using fluorescein-labeled tomato lectin (green; n ¼ 5 for each group). The total area of the vascular bed was visualized by immunostaining for CD31 (red; n ¼ 5 for each group). The fraction perfused area was expressed as perfused area divided by total vascular area for each field. (d) Apoptotic index of D2F2/E2 tumor cells as determined by immunostaining for activated caspase-3 (n ¼ 5 for each group). *Po0.05, **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Immunostaining, Marker, Labeling

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 4 T cells are not mediators of the effector functions of the DLL4 vaccine. (a) Mouse DLL4-specific T cell responses following immunization by protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP) were assessed by IFN-g ELISpot by stimulation of splenocytes with mouse DLL4 peptides or recombinant mouse extracellular portion of DLL4 (n ¼ 5 for each group). Recombinant carcinoembryonic antigen protein and carcinoembryonic antigen peptides were used as negative controls. The polyclonal stimulant concanavalin A (conA) was used as a positive control. The experiment was repeated three times. (b) Splenocyte activity in mice following immunization by protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP) at the time of sacrifice after tumor challenge was assessed by IFN-g ELISpot without antigen-specific stimulation (vector, n ¼ 9; protocol 1, n ¼ 8; protocol 2, n ¼ 5). (c) Depletion of CD4 þ or CD8 þ cells was initiated three days before tumor challenge by injection of anti-CD4 or anti-CD8 antibodies and verified by flow cytometric analysis of peripheral blood 5 days after injection of antibodies. Tumor growth curve from vector-immunized mice was duplicated from Figure 2c, which depicts an experiment performed simultaneously to the T cell depletion experiment. (d) Growth of orthotopically implanted D2F2/E2 tumors in control mice and in mice depleted of CD4 þ/CD8 þ cells following vaccination with DLL4 plasmid DNA (n ¼ 6 for all groups). **Po0.01, ***Po0.001; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Enzyme-linked Immunospot, Recombinant, Positive Control, Activity Assay, Plasmid Preparation, Injection, Control

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 5 Tumor growth suppression in DLL4-vaccinated mice is mediated by induction of anti-DLL4 antibodies. (a) The presence of anti-mouse DLL4 antibodies in the serum of DLL4-immunized mice was analyzed by ELISA. The figure depicts a representative analysis for one out of four mice analyzed. (b) IsolectinB4 staining of wholemounted P5 mouse retinas from mice treated with immune serum from vector or DLL4 immunized mice according to protocol 1 (3 ID þ EP; n ¼ 4 for each group). Arrowheads indicate endothelial cells morphologically identified as tip cells. (c) Growth of orthotopically implanted D2F2/E2 tumors in mice treated with serum from mice immunized with vector (n ¼ 12) or DLL4 plasmid DNA (n ¼ 16) according to protocol 1 (3 ID þ EP). The experiment was repeated twice with similar results. **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Plasmid Preparation

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 6 No evidence for liver toxicity or delayed wound healing following immunization with the DLL4 vaccine. (a) Hematoxylin and eosin staining of tissue sections of liver from mice that initiated the immunization procedure with empty vector or the DLL4 vaccine 5 months earlier. (b) Rate of healing expressed as % closure of wounds inflicted to mice immunized with empty vector or the DLL4 vaccine (n ¼ 8 mice per group, two wounds per mouse). *Po0.05, repeated-measures ANOVA.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Staining, Plasmid Preparation