antibody against egfl7  (R&D Systems)

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC enhances EGFL7 level by mediating histone H3 acetylation of EGFL7 promoter. a : URRCC subnetwork according to the result of Target mRNA PCR Array. Genes colored in blue are down-regulated genes after that cells were transfected with sh-URRCC. Genes colored in red are up-regulated genes after that cells were transfected with sh-URRCC. The size of the gene round represents the fold change. b : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with sh-control or sh-URRCC. c : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with mock or oe-URRCC. d : EGFL7 expression in ccRCC and normal samples from TCGA KIRC dataset. e : Representative EGFL7 IHC staining of ccRCC tissues compared to paired normal renal tissues (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). f and g : Representative EGFL7 IHC staining of xenograft tumors from sh-control, sh-URRCC, mock, and oe-URRCC groups (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). h : qRT-PCR and WB analysis of EGFL7 in A498 and OSRC-2 cells treated with DMSO or trichostatin A (TSA) (50 nM or 100 nM) for 72 h ( n = 3). i : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. j : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the GAPDH promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. k : ChIP analyses of OSRC-2 cells transfected with mock or oe-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. l : EGFL7 promoter region was enriched with H3K27Ac histone mark presented with UCSC data. m : WB analysis of H3K27ac and total H3 in A498 and OSRC-2 cells treated with DMSO or TSA, β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Transfection, Quantitative RT-PCR, Control, Expressing, Immunohistochemistry

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC is associated with EGFL7/P-AKT/FOXO3 signaling pathway. a and b : WB analysis of T-AKT, P-AKT, and FOXO3 in URRCC-overexpressing or URRCC-inhibited A498 and OSRC-2 cells compared with sh-control or mock group, β-actin was used as a loading control. c : CCK8 assays of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. d : Representative images and the numbers of invasive cells per high-power field reduced after the transfection with mock/oe-URRCC and si-NC/si-EGFL7 in A498 cells. e : WB analysis for EGFL7, T-AKT, P-AKT, and FOXO3 protein levels of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Control, Transfection

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: A Schematic Diagram of LncRNA-URRCC-Based Signaling Pathway in ccRCC cells proliferation and invasion. LncRNA-URRCC upregulates AKT signaling by directly targeting EGFL7 via mediating histone H3 acetylation of EGFL7 promoter, and then inducing cell growth and invasion. AKT signaling downstream FOXO3 in turn downregulates URRCC by directly binding to its promoter

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Binding Assay

Journal: Biomedical Reports

Article Title: Erythropoietin attenuates hyperoxia-induced lung injury by upregulating epidermal growth factor-like domain 7 in newborn rats

doi: 10.3892/br.2016.820

Figure Lengend Snippet: mRNA expression levels of (A) EGFL7, (B) Bcl-2 and (C) Bax were measured using the 2 −ΔΔCq method. Values are presented as the means ± standard deviation (n=8). *P<0.05 vs. time-matched hyperoxia group; **P<0.01 vs. time-matched hyperoxia group. # P<0.01 vs. control group. EGFL7, epidermal growth factor-like domain 7; Bcl-2, B-cell lymphoma 2; Bax, bcl-2-like protein 4; BPD, bronchopulmonary dysplasia.

Article Snippet: The membranes were subsequently incubated with primary rabbit polyclonal antibodies against EGFL7 (cat. no. 19291-1-AP; dilution, 1:500; ProteinTech Group, Inc., Chicago, IL, USA), Bax (cat. no. ab182733; dilution, 1:1,000; Epitomics; Abcam, Cambridge, USA), Bcl-2 (cat. no. BS1511; dilution, 1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA) and β-actin (cat. no. BS1002; dilution, 1:2,000; Bioworld Technology, Inc.) overnight at 4°C.

Techniques: Expressing, Standard Deviation, Control

Journal: Biomedical Reports

Article Title: Erythropoietin attenuates hyperoxia-induced lung injury by upregulating epidermal growth factor-like domain 7 in newborn rats

doi: 10.3892/br.2016.820

Figure Lengend Snippet: (A) Protein expression of EGFL7, Bcl-2 and Bax in lung tissue samples from each group was examined by western blot analysis. (B-D) Quantification of western blot analysis measured by the mean ratios of EGFL7/β-actin, Bcl-2/β-actin and Bax/β-actin. β-actin served to verify equivalent loading. Values are the means ± standard deviation (n=8). *P<0.05 and **P<0.01 vs. time-matched hyperoxia group; # P<0.01 vs. control group. EGFL7, epidermal growth factor-like domain 7; Bcl-2, B-cell lymphoma 2; Bax, bcl-2-like protein 4.

Article Snippet: The membranes were subsequently incubated with primary rabbit polyclonal antibodies against EGFL7 (cat. no. 19291-1-AP; dilution, 1:500; ProteinTech Group, Inc., Chicago, IL, USA), Bax (cat. no. ab182733; dilution, 1:1,000; Epitomics; Abcam, Cambridge, USA), Bcl-2 (cat. no. BS1511; dilution, 1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA) and β-actin (cat. no. BS1002; dilution, 1:2,000; Bioworld Technology, Inc.) overnight at 4°C.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: PLoS ONE

Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

doi: 10.1371/journal.pone.0012156

Figure Lengend Snippet: A . Top: schematic representation of the distal region of the mouse chromosome 2. The region encompassing the VE-statin/egfl7 gene is indicated with an arrow. Bottom: distribution of the genes along this region of the chromosome represented to scale, arrowheads indicate the orientation of the genes. B . Scaled schematic representation of the VE-statin/egfl7 gene. Arrows indicate the previously identified transcription start sites , exons are represented as numbered black boxes. C . Expression levels of notch1 , VE-statin/egfl7 , and agpat2 were measured in cultured L929 and 3T3 fibroblasts (white bars), and in 1G11, H5V, EOMA, and MS1 endothelial cells (black bars) after total RNA isolation and RT-qPCR. Levels of each transcripts were normalized to that of GAPDH measured in the same sample and represented as fold over mean levels in L929 cells set to 1. Y-axis scales are Log10 representations. *** p<0.001, ** p<0.01, * p<0.05.

Article Snippet: Membranes were incubated in PBS, 0.05% Tween-20, 5% non-fat dry milk (Soleil d'Agadir, Lille) for 3h at room temperature and incubated with antibodies directed against VE-statin/egfl7 (1/500, sc-66874), Erg (1/5000, sc-354x, Santa-Cruz), Fli-1 (1/1000, sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (1/2500, sc-9008x, Santa-Cruz), GATA-4 (1/2500, sc-1237x, Santa-Cruz), or beta-actin (1/1000, sc-1615) in PBS, 0.05% Tween-20, 5% non-fat-dry milk overnight at 4°C under constant mixing, rinsed three times 10 min with PBS, 0.25% Tween-20 at room temperature and further incubated with an horseradish peroxydase-coupled anti-goat, mouse, or rabbit antibody (1/10000, A9452-1VL Sigma, NA931VS, or NA934VS GE-Healthcare, respectively) in PBS, 0.05% Tween-20, 5% non-fat dry milk, rinsed three times in PBS, 0.25% Tween-20 and immunocomplexes were revealed using the Western Lightning-ECL kit (Perkin-Elmer) after exposure to Hyperfilm ECL (GE-Healthcare).

Techniques: Expressing, Cell Culture, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

doi: 10.1371/journal.pone.0012156

Figure Lengend Snippet: Top. Levels of acetylated-histone H3 along the VE-statin/egfl7 gene were quantified using chromatin immunoprecipitation of 1% formaldehyde-treated cultures of H5V endothelial (•, black bars), L929 (□, white bars), and 3T3 (Δ, grey bars) fibroblast DNA followed by sonication, immunoprecipitation of the DNA-protein complexes using an acetyl Histone-H3 antibody (06-599, Millipore), DNA purification and semi-quantitative PCR analysis performed in order to amplify various locations along the promoter represented along the x-axis. Quantities are relative to the diluted INPUT mean value set to 1. Acetylation levels were low in all cells in the regions located ahead of the transcription start -1b, they were elevated only in endothelial cells thereafter. Inset: Acetylated-histone H3 levels of the negative control β-globin and the positive control VE-cadherin gene promoters taken as non- and highly-expressed genes in endothelial cells, respectively, and assessed in similar conditions. Bottom. Scale schematic representation of the mouse VE-statin/egfl7 promoter and gene, scaled according to the x-axis in A. Arrows indicate the previously identified transcription start sites , exons are represented as numbered black boxes.

Article Snippet: Membranes were incubated in PBS, 0.05% Tween-20, 5% non-fat dry milk (Soleil d'Agadir, Lille) for 3h at room temperature and incubated with antibodies directed against VE-statin/egfl7 (1/500, sc-66874), Erg (1/5000, sc-354x, Santa-Cruz), Fli-1 (1/1000, sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (1/2500, sc-9008x, Santa-Cruz), GATA-4 (1/2500, sc-1237x, Santa-Cruz), or beta-actin (1/1000, sc-1615) in PBS, 0.05% Tween-20, 5% non-fat-dry milk overnight at 4°C under constant mixing, rinsed three times 10 min with PBS, 0.25% Tween-20 at room temperature and further incubated with an horseradish peroxydase-coupled anti-goat, mouse, or rabbit antibody (1/10000, A9452-1VL Sigma, NA931VS, or NA934VS GE-Healthcare, respectively) in PBS, 0.05% Tween-20, 5% non-fat dry milk, rinsed three times in PBS, 0.25% Tween-20 and immunocomplexes were revealed using the Western Lightning-ECL kit (Perkin-Elmer) after exposure to Hyperfilm ECL (GE-Healthcare).

Techniques: Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, DNA Purification, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

doi: 10.1371/journal.pone.0012156

Figure Lengend Snippet: A . H5V endothelial (black bars) and L929 fibroblast (right, white bars) were transfected with the pGL3basic luciferase reporter vector (Ctrl) or pGL3basic in which −12969/+38 VE-statin/egfl7 promoter region or 5′ deletions of it were inserted. These reporters (80 fmoles) were transfected together with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Numbers associated with the black bars represent the calculated endothelial/fibroblast ratio of activity for the corresponding construct. Left: scaled schematic representation of the constructs, names are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. The experiment is representative of a set of at least three experiments performed in similar conditions. Constructions were designed so that the luciferase gene was placed within exon-1b because initial experiments showed that placing it where the coding sequence starts in exon-3 resulted in no detectable activity (not shown). ** p<0.01, * p<0.05. B . H5V endothelial cells (black bars) or 3T3 fibroblasts (white bars) were transfected with 80 fmoles of pGL3promoter (Ctrl, Promega), −8409/−7563SV40Luc, −7563/−8409-SV40Luc, or −7770/−7563-SV40Luc and with 54 fmoles of pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Results are displayed as in Figure 3A. The −8409/−7563 fragment is active in endothelial cells regardless of orientation, the most active sequence in this fragment corresponds to region A (−7770/−7563) which shows an endothelial/fibroblast ratio similar to the reporter containing the whole −8409/−7688 region.

Article Snippet: Membranes were incubated in PBS, 0.05% Tween-20, 5% non-fat dry milk (Soleil d'Agadir, Lille) for 3h at room temperature and incubated with antibodies directed against VE-statin/egfl7 (1/500, sc-66874), Erg (1/5000, sc-354x, Santa-Cruz), Fli-1 (1/1000, sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (1/2500, sc-9008x, Santa-Cruz), GATA-4 (1/2500, sc-1237x, Santa-Cruz), or beta-actin (1/1000, sc-1615) in PBS, 0.05% Tween-20, 5% non-fat-dry milk overnight at 4°C under constant mixing, rinsed three times 10 min with PBS, 0.25% Tween-20 at room temperature and further incubated with an horseradish peroxydase-coupled anti-goat, mouse, or rabbit antibody (1/10000, A9452-1VL Sigma, NA931VS, or NA934VS GE-Healthcare, respectively) in PBS, 0.05% Tween-20, 5% non-fat dry milk, rinsed three times in PBS, 0.25% Tween-20 and immunocomplexes were revealed using the Western Lightning-ECL kit (Perkin-Elmer) after exposure to Hyperfilm ECL (GE-Healthcare).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct, Clone Assay, Sequencing

Journal: PLoS ONE

Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

doi: 10.1371/journal.pone.0012156

Figure Lengend Snippet: A . H5V endothelial cells were transfected with siRNA targeting fli-1, erg, or ets-1 ( A ), gata-2, or gata-4 ( B ) and cultured 48h before total RNA extraction and qPCR analysis of endogenous VE-statin/egfl7 expression. ** p<0.01, ns; not significant. Quantities are relative to mean value of the Ctrl siRNA set to 1. Down-regulation of Erg, Fli-1, gata-2 and gata-4 resulted in a significant decrease in VE-statin/egfl7 expression levels. Specificity of the siRNA: qPCR analysis of expression levels of the erg, fli-1 and ets-1 ( C ) and of gata-2 and gata-4 ( D ) genes in response to either siRNA. Levels were normalized to the mean value obtained in response to transfection to the Ctrl siRNA set to 1. All siRNA show a good specificity of inhibition toward their target and no significant side effects on the other studied genes of the same family. Of note, the siRNA targeting Ets-1 down-regulates Fli-1 as well, as expected since Ets-1 controls the fli-1 gene in endothelial cells .

Article Snippet: Membranes were incubated in PBS, 0.05% Tween-20, 5% non-fat dry milk (Soleil d'Agadir, Lille) for 3h at room temperature and incubated with antibodies directed against VE-statin/egfl7 (1/500, sc-66874), Erg (1/5000, sc-354x, Santa-Cruz), Fli-1 (1/1000, sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (1/2500, sc-9008x, Santa-Cruz), GATA-4 (1/2500, sc-1237x, Santa-Cruz), or beta-actin (1/1000, sc-1615) in PBS, 0.05% Tween-20, 5% non-fat-dry milk overnight at 4°C under constant mixing, rinsed three times 10 min with PBS, 0.25% Tween-20 at room temperature and further incubated with an horseradish peroxydase-coupled anti-goat, mouse, or rabbit antibody (1/10000, A9452-1VL Sigma, NA931VS, or NA934VS GE-Healthcare, respectively) in PBS, 0.05% Tween-20, 5% non-fat dry milk, rinsed three times in PBS, 0.25% Tween-20 and immunocomplexes were revealed using the Western Lightning-ECL kit (Perkin-Elmer) after exposure to Hyperfilm ECL (GE-Healthcare).

Techniques: Transfection, Cell Culture, RNA Extraction, Expressing, Inhibition

Journal: PLoS ONE

Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

doi: 10.1371/journal.pone.0012156

Figure Lengend Snippet: H5V endothelial cells were transfected with a control siRNA (siCtrl) of with siRNA targeting ets-1, fli-1, erg, gata-2, or gata-4, cultured for 48hr and analysed for protein expression by SDS-PAGE and Western-blotting. VE-statin/egfl7 accumulation (boxed) is impaired when cells were treated with siRNA targeting fli-1, erg and, and, to a slight extent, GATA-2, or when treated with a siRNA targeting VE-statin/egfl7 itself, used as positive control. Of note, there is a cross-regulation between gata-2 and fli-1, as already reported in hematopoietic stem cells . Specific bands are indicated by arrowheads. The Ets-1 band is distinguishable from a large non-specific band located immediately below .

Article Snippet: Membranes were incubated in PBS, 0.05% Tween-20, 5% non-fat dry milk (Soleil d'Agadir, Lille) for 3h at room temperature and incubated with antibodies directed against VE-statin/egfl7 (1/500, sc-66874), Erg (1/5000, sc-354x, Santa-Cruz), Fli-1 (1/1000, sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (1/2500, sc-9008x, Santa-Cruz), GATA-4 (1/2500, sc-1237x, Santa-Cruz), or beta-actin (1/1000, sc-1615) in PBS, 0.05% Tween-20, 5% non-fat-dry milk overnight at 4°C under constant mixing, rinsed three times 10 min with PBS, 0.25% Tween-20 at room temperature and further incubated with an horseradish peroxydase-coupled anti-goat, mouse, or rabbit antibody (1/10000, A9452-1VL Sigma, NA931VS, or NA934VS GE-Healthcare, respectively) in PBS, 0.05% Tween-20, 5% non-fat dry milk, rinsed three times in PBS, 0.25% Tween-20 and immunocomplexes were revealed using the Western Lightning-ECL kit (Perkin-Elmer) after exposure to Hyperfilm ECL (GE-Healthcare).

Techniques: Transfection, Control, Cell Culture, Expressing, SDS Page, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

doi: 10.1371/journal.pone.0012156

Figure Lengend Snippet: H5V cell cultures were treated with 1% formaldehyde, sonicated in order to produce 1000 bp average size DNA fragments and immunoprecipitation of the DNA-protein complexes was performed using antibodies against HA.11 (irrelevant Ab, MMS-101R, Convance), Erg (sc-354x, Santa-Cruz), Fli-1 (sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (sc-9008x, Santa-Cruz), or GATA-4 (sc-1237x, Santa-Cruz) antibody. DNA was then purified and analyzed by semi-quantitative PCR on the −252/+38 promoter region of the endogenous VE-statin/egfl7 gene. INPUT; 3.5% of total DNA, No Ab; no antibody added, Irrel Ab; irrelevant IgG added. Levels were measured by semi-quantitative PCR using oligonucleotides that span the −252/+38 region of the gene. Data are expressed as 2 −ΔCt where ΔCt = Ct of sample – Ct of INPUT. The experiment is representative of a set of three performed in similar conditions. *** p<0.001, * p<0.05.

Article Snippet: Membranes were incubated in PBS, 0.05% Tween-20, 5% non-fat dry milk (Soleil d'Agadir, Lille) for 3h at room temperature and incubated with antibodies directed against VE-statin/egfl7 (1/500, sc-66874), Erg (1/5000, sc-354x, Santa-Cruz), Fli-1 (1/1000, sc-356x, Santa-Cruz), Ets-1 (sc-111x, Santa Cruz), GATA-2 (1/2500, sc-9008x, Santa-Cruz), GATA-4 (1/2500, sc-1237x, Santa-Cruz), or beta-actin (1/1000, sc-1615) in PBS, 0.05% Tween-20, 5% non-fat-dry milk overnight at 4°C under constant mixing, rinsed three times 10 min with PBS, 0.25% Tween-20 at room temperature and further incubated with an horseradish peroxydase-coupled anti-goat, mouse, or rabbit antibody (1/10000, A9452-1VL Sigma, NA931VS, or NA934VS GE-Healthcare, respectively) in PBS, 0.05% Tween-20, 5% non-fat dry milk, rinsed three times in PBS, 0.25% Tween-20 and immunocomplexes were revealed using the Western Lightning-ECL kit (Perkin-Elmer) after exposure to Hyperfilm ECL (GE-Healthcare).

Techniques: Sonication, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction