goat anti mouse egfl7  (R&D Systems)

Journal: Development (Cambridge, England)

Article Title: GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126

doi: 10.1242/dev.184218

Figure Lengend Snippet: EGFL7/miR-126 is a target of GATA2. (A) miR-126 was expressed in the dorsal aorta and in the LVV-ECs (arrow) of control embryos. (B) Expression of miR-126 was downregulated in the LVV-ECs (arrow) of E12.0 Lyve1-Cre;Gata2 f/f embryos. However, no obvious difference in miR-126 expression was observed in the dorsal aorta of mutants. Red dashed line indicates the endothelial layer of the lymph sac. (C) EGFL7 was expressed in the mesenteric arteries, veins and lymphatic vessels of E18.5 control embryos. The strongest expression of EGFL7 was observed in LVs (arrow). (D) Expression of EGFL7 was dramatically downregulated in the mesenteric lymphatic vessels of mice lacking GATA2 in LECs. Also, notice the absence of LVs in the mutant. (E) ChIP revealed that GATA2 strongly associates with the promoter element of the EGFL7/miR-126 locus. The top gel shows PCR performed using primers flanking the GATA2-binding site. The lower gel shows PCR performed using primers for a non-specific site. The graph compares qPCR signals generated by primers flanking the GATA2-binding site. A, artery; DA, dorsal aorta; L, lymphatic vessel; LS, lymph sac; V, vein. (A,B) n =3 embryos and 6 LVV complexes per genotype; (C,D) n =3 embryos per genotype; (E) n =4. ** P <0.01. Scale bars: 250 μm (A,B); 200 μm (C,D).

Article Snippet: Primary antibodies for immunohistochemistry were: rabbit anti-PROX1 (11-002, Angiobio), goat anti-human PROX1 (AF2727, R&D Systems), sheep anti-mouse FOXC2 (AF6989, R&D Systems), goat anti-mouse VEGRF3 (AF743, R&D Systems), rat anti-mouse CD31 (553370, BD Pharmingen), goat anti-mouse ITGA9 (AF3827, R&D Systems), rat anti-mouse VE-cadherin (550548, BD Pharmingen), hamster anti-mouse PDPN (127401, Biolegend), rat anti-mouse ITGA5 (553319, BD Pharmingen), goat anti-mouse GATA2 (AF2046, R&D Systems), rabbit anti-mouse CX37 (40-4200, Life Technologies), rabbit anti-mouse LAMA5 (Ab11575, Abcam), rabbit anti-human fibronectin (ab2413, Abcam), goat anti-human ANGPT2 (AF623, R&D Systems), goat anti-mouse EGFL7 (AF3089, R&D Systems), rabbit anti-mouse CLDN5 (34-1600, Thermo Fisher Scientific), rabbit anti-mouse LYVE-1 (11-034, Angiobio).

Techniques: Control, Expressing, Mutagenesis, Binding Assay, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Comparison of clinical and molecular characteristics by EGFL7 -expresser status of younger adult patients (age <60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Prognostic significance of EGFL7 in younger and older CN-AML patients. (A and B) Impact of EGFL7 expression levels on DFS of younger (age <60 y) (A) and older (age ≥60 y) (B) adult patients. (C) DFS according to EGFL7 risk group in older CN-AML patients. The favorable risk group comprised patients with EGFL7 low expression/high methylation; the unfavorable risk group comprised the remaining patients (high expression/low methylation, high expression/high methylation, low expression/low methylation). The median values of EGFL7 expression and EGFL7 promoter methylation were used as cutoffs.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing, Methylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Prognostic significance of EGFL7 in younger and older CN-AML patients. (A and B) The association of EGFL7 expression levels with OS and EFS of younger adult patients (age <60 y) (A) and older patients (age ≥60 y) (B). (C) OS and EFS according to EGFL7 risk group in older CN-AML patients. The favorable risk group was comprised of patients with EGFL7 low expression/high promoter methylation; the unfavorable risk group included the remaining patients (high expression/low promoter methylation, high expression/high promoter methylation, low expression/low promoter methylation). The median values of EGFL7 expression and EGFL7 promoter methylation were used as the high/low cut points.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing, Methylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Treatment outcomes according to EGFL7 expression in 374 younger adult patients (age <60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Comparison of clinical and molecular characteristics by EGFL7 -expresser status of older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Treatment outcomes according to EGFL7 expression in 198 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Univariable models for expression of EGFL7 and miR-126 and associations with outcome in 300 younger adults and 171 older patients with CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Treatment outcomes according to EGFL7 risk group in 126 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Multivariable analyses of outcome according to the EGFL7 risk group in 126 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Multivariable analyses of event-free survival according to the EGFL7 risk group in 126 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Cytogenetic and molecular characteristics of the leukapheresis samples from AML patients that were profiled for EGFL7 expression and were used in functional studies

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing, Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 is up-regulated in human and mouse AML cells. (A) NBM samples from healthy donors (n = 3) were compared with leukapheresis samples of AML patients (ptAML, n = 11). EGFL7 levels were measured in AML samples by real-time RT-PCR, and the results were normalized to β-ACTIN RNA levels. (B) Mean ± SD of EGFL7 mRNA expression levels between NBM and AML in aggregate. *P < 0.05. (C) EGFL7 protein levels in human NBM and leukapheresis samples of AML patients were determined by immunoblotting with GAPDH as loading control. (D) Normal BM from WT mice (n = 4) was compared with murine AML blasts of the MllPTD/WTFlt3ITD/WT mouse model (mAML, n = 4) for the detection of mouse Egfl7 mRNA by RT-PCR with β-Actin as internal control. (E) Mean ± SD of Egfl7 mRNA between murine NBM and murine AML blasts, in aggregate. **P < 0.01. (F) Mouse Egfl7 protein levels in WT murine controls (n = 4) and murine AML blasts from the MllPTD/WTFlt3ITD/WT mouse model (mAML) (n = 4) were assessed by immunoblotting using Gapdh as loading control. (G) Immunohistochemistry of Egfl7 in NBM of WT (n = 3) control mice vs. BM from MllPTD/WTFlt3ITD/WT leukemic mice (n = 3) using an Egfl7-specific antibody or no antibody controls. (Original magnification: 100×; Insets with same areas across the samples are magnified at same strength.) (H) Percent of Egfl7+ cells in NBM of WT mice vs. BM from MllPTD/WTFlt3ITD/WT leukemic mice. ****P < 0.0001. (I and J) EGFL7 mRNA (I) and EGFL7 protein (J) expression levels in four human AML cell lines (EOL1, OCI-AML3, MV4-11, and Kasumi-1). The relative expression of EGFL7 mRNA was measured with real-time RT-PCR normalized to β-ACTIN. For immunoblotting, β-Tubulin was used as loading control.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 is a secreted protein and is increased in the serum of some AML patients. (A) Blasts of AML patients (ptAML, n = 3) were cultured in SFEM medium + 10% FBS supplemented with cytokines for 24 h. The EGFL7 protein level in the cell-culture supernatant was detected by ELISA and was compared with that in medium from wells without cultured cells. **P < 0.01, *P < 0.05. (B) Blasts of AML patients (n = 3) were cultured in SFEM medium + 10% FBS supplemented with cytokines for 24 h. The EGFL7 protein in the cell-culture supernatant was assessed by immunoblotting with rEGFL7 as a positive control and medium from wells without cultured cells as a negative control. Ponceau S staining shows the loading control for protein. (C) EGFL7 protein levels in sera from normal healthy controls (sN, n = 6) and AML patients (sAML, n = 6) were determined by the EGFL7 ELISA kit. SFEM medium alone and 10% FBS serve as blank controls. *P < 0.05, **P < 0.01. (D) An equal volume of serum from AML patients (sAML, n = 12) or normal healthy donors (sN, n = 6) was subjected to the separation of exosomal vs. nonexosomal eluant using the ExoQuick kit (System Biosciences). EGFL7 protein levels in both isolated exosomes and the supernatant were determined by immunoblotting. SFEM containing 10% FBS served as a negative control, and rEGFL7 was used as a positive control. Ponceau S staining shows the loading of proteins.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Negative Control, Staining, Control, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 stimulates the proliferation of human and mouse AML cells. (A and B) BM cells from WT (A) and MllPTD/WTFlt3ITD/WT (B) mice were treated without (Unstim) or with 0.1, 0.25, or 0.5 μM rEgfl7 in Iscove’s Modified Dulbecco’s Medium (IMDM) + 2% BSA for 24, 48, 72, or 96 h. At the indicated time points, the number of viable cells was determined by Trypan blue dye exclusion assay. Each condition was repeated in triplicate. *P < 0.05, **P < 0.01; NS, not significant. (C) Blasts of the indicated AML patients (20,000 cells) were mixed with methylcellulose medium in the absence or presence of 0.25 μM rEGFL7 and were plated onto 2-cm dishes for 10 d. Colonies with more than 50 cells were enumerated using a light microscope. Each condition for each patient (n = 4) was plated in triplicate; **P < 0.01, ***P < 0.001. (D) Kasumi-1 cells were stimulated with 100 nM rEGFL7 for 4 h in RPMI1640 with 10% FBS. Cell proliferation was assessed using APC-BrdU/7AAD staining coupled with flow cytometry; *P < 0.05. (E) Kasumi-1 cells (2,500 cells) were mixed with methylcellulose medium in the absence or presence of 100 nM recombinant human EGFL7 and were scored after 10 d. Each condition was plated in triplicate in three independent experiments. *P < 0.05. (F) Blasts from AML patients (n = 4) were cultured in SFEM + 2% BSA in the absence or presence of 0.25 μM rEGFL7 for 20 min. Total proteins were extracted for immunoblotting of pAKT-S473 and total AKT. GAPDH was used as loading control. (G) AML blasts from MllPTD/WTFlt3ITD/WT mice (n = 3) were cultured in IMDM medium + 2% BSA in the absence or presence of 0.25 μM rEgfl7 for 20 min. Total proteins were extracted for immunoblotting of pAkt-S473 and total Akt. Gapdh was used as loading control. (H) Exponentially growing Kasumi-1 cells were starved in serum-free RPMI1640 medium for 1 h, followed by the addition of 100 nM recombinant EGFL7 for 5 min. Total proteins were extracted for immunoblotting of pAKT-S473, total AKT, and GAPDH.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Modification, Exclusion Assay, Light Microscopy, Staining, Flow Cytometry, Recombinant, Cell Culture, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 inhibition results in decreased human AML cell growth without affecting normal hematopoietic cells. (A) Blasts from AML patients were cultured in SFEM with 10% FBS in the presence of 50 μg/mL of normal human IgG or anti-EGFL7 (@E7) antibody for 1 h. Total proteins were extracted for immunoblotting of pAKT-S473 and total AKT. GAPDH was used as loading control. (B) Human primary blasts (400,000) from AML patients (n = 4) were treated with 2, 10, 50, or 250 μg/mL of IgG control or anti-EGFL7 antibody in SFEM containing 10% FBS and cytokines for 2 h. Twenty thousand cells were plated in triplicate in methylcellulose medium and scored after 14 d of culture for mean ± SD colony numbers. *P < 0.05, **P < 0.01. (C) Forty-eight hours after IgG control vs. anti-EGFL7 antibody treatment (50 μg/mL) of AML cell lines, apoptosis was evaluated by Annexin V/7AAD staining, (D and E) Cell proliferation was measured using BrdU incorporation (D), and differentiation analysis was evaluated by CD11b expression (E). CD11b is depicted as the ratio of the CD11b expression value of the examined sample to the CD11b expression value of the corresponding IgG-treated control. *P < 0.05, **P < 0.01. (F) CD34+ CB cells from four different donors were plated in methylcellulose medium in the presence of increasing concentrations (2, 10, 50, or 250 μg/mL) of human IgG or anti-EGFL7 and were scored after 14 d of culture. Along with total number of colonies, colony types [erythroid burst-forming units (BFU), granulocyte/monocyte (GM), or granulocyte/erythrocyte/monocyte/megakaryocyte colonies (GEMM)] were enumerated also.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Inhibition, Cell Culture, Western Blot, Control, Staining, BrdU Incorporation Assay, Expressing