hm1099  (R&D Systems)

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A–D ) Immunohistochemistry on cross sections of hearts. Panels show the outer part of the wall of the left ventricle. ( A ) EphB4 is expressed in veins, venules (white arrowheads) and small capillaries (blue arrowheads) in the adult heart ventricle. ( B ) Concomitant expression of GFP shows that ephrin-B2 is expressed in the small capillaries of the ventricular wall (arrowheads) and in arteries. a, artery; v, vein. ( C ) Cdh5 CreERT2 R26-mTmG double heterozygous mice show GFP expression throughout the cardiac endothelium at 4 weeks after tamoxifen induction. ( D ) EphB4 protein expression is abolished in Ephb4 ∆EC cardiac veins, venules (white arrowheads) and capillaries (blue arrowheads). ( E ) Western blot analysis of total heart lysate at 12 weeks confirms strongly reduced EphB4 expression in Ephb4 ∆EC mutants. Molecular weight marker (kDa) is indicated. a, artery; v, vein.

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunohistochemistry, Expressing, Western Blot, Molecular Weight, Marker

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) Freshly dissected Ephb4 ∆EC and littermate control hearts at 12 weeks of age. Heart weight and heart weight/tibia length index (HW/TL) ratio are increased in Ephb4 ∆EC mutants, whereas body weight remains unchanged. N = 5 for control and N = 8 for Ephb4 ∆EC . ( B ) Immunohistochemistry on cross sections of control and Ephb4 ∆EC hearts at 12 weeks. Panels show the inner part of the left ventricular wall with significantly increased cardiomyocyte relative area in Ephb4 ∆EC samples. N = 4 for both genotypes. ( C ) Echocardiography analysis of 12 week-old animals. Ejection fraction (EF) is significantly reduced in mutant mice while left ventricle diastolic and systolic volumes are increased. lv, left ventricle. N = 10 for control and N = 6 for Ephb4 ∆EC ( D ) CMRI sections of hearts showing four-chamber (left) and short axis (right) views of control and Ephb4 ∆EC at 12 weeks of age. Arrowheads indicate the ventricular septum and the wall of the left ventricle. rv, right ventricle; lv, left ventricle. Ventricular septum thickness and left ventricular (lv) mass are significantly reduced in Ephb4 ∆EC mice, whereas right ventricular (rv) mass remains unchanged. N = 9 for control and N = 11 for Ephb4 ∆EC . Data represented as mean ± s.e.m. P values calculated by unpaired two-tailed t test with Welch’s correction. ns, not significant. Figure 1—source data 1. Source data for .

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunohistochemistry, Mutagenesis, Two Tailed Test

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A–C ) Immunostaining of cross sections through 12 week-old control and Ephb4 ∆EC hearts. Confocal images show the outer ( A, C ) and inner part ( B ) of the left ventricle. ( A ) Vascular density, measured by number of branching points, is significantly reduced in Ephb4 ∆EC samples. N = 3 for control and N = 6 for Ephb4 ∆EC . ( B ) Pericyte coverage is reduced in Ephb4 ∆EC hearts (arrowheads mark affected capillaries). N = 3 per genotype. ( C ) Presence of microhemorrhages (arrowheads mark erythrocytes in the mutant myocardium). ( D ) Electron micrographs of control and Ephb4 ∆EC capillaries. Bottom images are higher magnifications of boxed areas in upper panels. White arrowheads indicate accumulation of caveolar vesicles at the mutant endothelial basolateral membrane (center) and thrombocytes in a vascular rupture (right). Yellow arrowheads indicate mitochondrial glycogen accumulation. Erythrocytes (er), thrombocytes (th), cardiomyocytes (cm) and endothelial cells (ec) are indicated. Data represented as mean ± s.e.m. P values calculated by unpaired two-tailed t test with Welch’s correction. Figure 2—source data 1. Source data for .

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunostaining, Mutagenesis, Two Tailed Test

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) TUNEL assay in Ephb4 ∆EC hearts. There is no detectable cell death in mutant ECs of the ventricular wall. ( B ) Hypoxia assay using Pimonidazole shows that Ephb4 ∆EC hearts are not hypoxic. Hearts were compared to a known hypoxic tissue ( C ), namely bone marrow in long bone, as a positive control. Arrowheads indicate positive signal (red).

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: TUNEL Assay, Mutagenesis, Positive Control

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) Immunohistochemistry on for Collagen I (ColI, green) and Collagen IV (ColIV, green) in transverse sections of control and Ephb4 ∆EC heart showing both ventricles. Arrowheads mark normal signal. ECs are stained by isolectin B4 (IB4, blue), nuclei by DAPI (white). ( B ) Masson’s Gold Trichrome staining on cross sections of hearts. No fibrosis is observed. lv, left ventricle, rv, right ventricle.

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunohistochemistry, Staining

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A, B ) Immunostaining on cross sections of the outer wall of the left ventricle. ( A ) Presence of microhemorrhages in the myocardium of Ephb4 ∆EC mice at 10 weeks. ( B ) Presence of Ter119+ red blood cells (red) in the myocardium of Efnb2 ∆EC mice at 12 weeks. Arrowheads indicate erythrocytes outside of blood vessels.

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunostaining

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A–D ) Immunohistochemistry on longitudinal sections of control and Ephb4 ∆EC gastrocnemius at 12 weeks of age. ( A ) EphB4 and ephrin-B2 (GFP in Efnb2::GFP reporter line) are expressed in capillaries (arrowheads). ( B ) Vascular density, measured by number of branching points, is not changed in Ephb4 ∆EC gastrocnemius. ( C ) Pericyte coverage and ( D ) myocyte (M) relative area is not reduced in Ephb4 ∆EC gastrocnemius. N = 3 for both genotypes. Data represented as mean ± s.e.m. P values calculated by unpaired two-tailed t test with Welch’s correction. ns, not significant. Figure 4—source data 1. Source data for .

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunohistochemistry, Two Tailed Test

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A, B ) Immunostaining on sections (150 µm thick) of the right kidney of 12 week-old Ephb4 ∆EC mutants and control littermates. No significant changes in kidney vasculature are visible after staining of CD31 ( A ) or ICAM ( B ) ( B ), green) and DAPI (nuclei, magenta). ( C ) Immunostaining of CD31 (green) in the sections (150 µm thick) from the Ephb4 ∆EC and control right median liver lobe does not reveal any overt differences in the hepatic vasculature. DAPI (nuclei, magenta).

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunostaining, Staining

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) Immunohistochemistry of lymphatic vessel in the intestinal mesentery. EphB4 immunosignal is lost in lymphatic vessels (arrowheads) but maintained in Ephb4 ∆LEC veins (arrows). ( B ) Control and Ephb4 ∆LEC hearts at 12 weeks of age. Heart weight and heart weight/tibia length index (HW/TL) are unchanged in Ephb4 ∆LEC mutants. N = 5 for control and N = 3 for Ephb4 ∆LEC . ( C ) Immunostaining of the inner part of the wall of the left ventricle. Cardiomyocyte (CM) relative area remains unaffected in Ephb4 ∆LEC hearts. N = 5 for control and N = 3 for Ephb4 ∆LEC . Data represented as mean ± s.e.m. P values calculated by unpaired two-tailed t test with Welch’s correction. ns, not significant.

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Immunohistochemistry, Immunostaining, Two Tailed Test

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A–F ) Global gene expression analysis of Ephb4 ∆EC ventricles by RNA sequencing at 12 weeks. ( A ) MA plot representing the 529 differentially expressed genes (p<0.05) including 268 upregulated genes (red) and 261 downregulated genes (blue). ( B–F ) Representation of the ten most significant functional categories in each group revealed by gene ontology analysis using the Enrichr data base. Graphs represent Enrichr combined score that combines P value and Z score. Numbers in brackets represent the number of differentially expressed genes in the corresponding category. ( B ) Human disease enriched terms according to the Online Mendelian Inheritance in Man (OMIM). Biological processes upregulated ( C ) and downregulated ( D ) in Ephb4 ∆EC ventricles. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways upregulated ( E ) and downregulated ( F ) in Ephb4 ∆EC ventricles. HCM, hypertrophic cardiomyopathy; DCM, dilated cardiomyopathy. Figure 5—source data 1. RNA-seq analysis of Ephb4 ∆EC and control mouse heart ventricles. The table indicates all the transcripts significantly upregulated and downregulated in Ephb4 mutant hearts. Figure 5—source data 2. Global proteome analysis of Ephb4 ∆EC and control mouse heart ventricles. The table indicates all the proteins significantly upregulated and downregulated in Ephb4 mutant hearts.

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Expressing, RNA Sequencing Assay, Functional Assay, Mutagenesis

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) Western blot analysis of HUVECs transfected with siControl , siEPHB4 or siCAV1 , as indicated. Knockdown cells showed a reduction of CAV1 and pCAV1 protein levels. N = 3 for all treatments. ( B ) Src input total cell lysate (TL) and tyrosine phosphorylation of immunoprecipitated (IP) Src in siRNA-treated HUVECs. Bottom panels show levels of CD36 and GAPDH (loading control). N = 3 for all treatments. ( C ) Quantitation of immunoblots for levels of EphB4, CAV1, pCAV1, CD36, pSrc/Src and ratio of pSrc/Src/ input. N = 3 for all treatments. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. ( D ) Confocal images of siRNA-transfected HUVECs stained with CAV1 or pCAV1 (red), GM130 (green), VE-Cadherin (CDH5; white) and nuclei (DAPI; blue). Bottom panels show surface CD36 (red), Phalloidin (white), and nuclei (DAPI; blue). ( E ) Quantitation of CAV1, pCAV1 and CD36 MFI per whole cell and of surface CD36 signal of immunostained HUVECs, as shown in ( D ). N = 3 experiments for CAV1 and pCAV1, in each of them 30 cells were quantified from three images (10 cells/ image). N=3 for CD36 surface staining, in which three images/experiment were quantified for all siRNA conditions. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. MFI, mean fluorescence intensity; ns, not significant. Figure 7—source data 1. Source data for . Figure 7—source data 2. Source data for .

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Western Blot, Transfection, Immunoprecipitation, Quantitation Assay, Staining, Fluorescence

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) Western blot analysis of CAV1 and pCAV1 levels in HUVECs stimulated with control human IgG/Fc (Ctrl Fc), ephrin-B2/Fc (B2/Fc) or EphB4/Fc (B4/Fc) (4 µg/ml, preclustered with 10µg/ml goat anti-human IgG) in combination with DMSO (vehicle control) or the Src inhibitor PP2. GAPDH is shown as loading control. ( B, F ) Quantitation of Western blots results (see A ) for pCAV1 together with the ratio pCAV1/CAV1 ( B ) and levels of CD36 ( F ). N = 3 for all treatments. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. ( C ) Confocal images of HUVECs after stimulation with Fc proteins in combination with DMSO or PP2. Stainings show pCAV1 (red), GM130 (green), CDH5 (white) and DAPI (blue). ( D ) Ratio of pCAV1/CAV1 in lysates from HUVECs treated with ephrin-B2/Fc in combination with DMSO (vehicle control) or the indicated inhibitors (LY294002, PP2 and U0126). N = 3 for all treatments. Data represented as mean ± s.e.m. P values calculated by unpaired two-tailed t test with Welch’s correction. ( E ) Quantitation of pCAV1 immunosignal per cell (as shown in C ) of HUVECs stimulated with Fc proteins along with inhibitor treatment. N = 3 experiments, in each of them 30 cells were quantified from three images (10 cells/ image) for all treatments. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA test. ( G ) Quantitation of normalized CAV1, pCAV1 and pCAV1/CAV1 in immunoblotted lysates from HUVECs treated with control siCtrl or siEPHB4 in combination with Ctrl Fc or ephrin-B2/Fc (B2/Fc). N = 3 for all conditions. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA. Norm. IntDen, normalized integrated density; ns, not significant. Figure 8—source data 1. Source data for . Figure 8—source data 2. Source data for . Figure 8—source data 3. Source data for . Figure 8—source data 4. Source data for .

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Western Blot, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: Endothelial EphB4 maintains vascular integrity and transport function in adult heart

doi: 10.7554/eLife.45863

Figure Lengend Snippet: ( A ) Confocal images of HUVECs transfected siControl , siEPHB4 and siCAV1 . Cells were fixed 30 min after exposure to BSA-555 (red) and immunostained with anti-CDH5 (white) antibody and DAPI (nuclei, blue). ( B ) Confocal images of siControl , siEPHB4 and siCAV1 HUVEC cells fixed after 30 min of treatment with BODIPY C 12 -500/510 (green) and immunostained for CDH5 (white). ( C ) Quantitation of BSA-555 (top) and BODIPY C 12 -500/510 (bottom) MFI per cell in siControl , siEPHB4 and siCAV1 HUVECs. Note reduced uptake of BSA and BODIPY in siEPHB4 and siCAV1 cells. N = 3 experiments, in each of them 30 cells were quantified from three images (10 cells/ image) for all knockdown conditions. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA test. ( D ) CD36 expression in 12 week-old control and Ephb4 ∆EC sectioned heart. Panels show details of the inner part of the left ventricle. CD36 immunosignal is reduced in mutant capillaries (arrowheads) but increased in the membrane of cardiomyocytes (arrows). MFI, mean fluorescence intensity. Figure 9—source data 1. Source data for .

Article Snippet: Primary antibodies: mouse anti-IB4-Biotin (Vector, B-1205, 1:25), WGA-tetramethylrhodamine (Invitrogen, W849, 1:100), chicken anti-GFP (2BScientific, GFP-1010, 1:100), rat anti-EphB4 (Hycult Biotechnology, HM1099, 1:100), goat anti-EphB4 (R and D Systems, AF446, 1:100) rat anti-Icam2 (BD Pharmingen, 553326, 1:100), mouse anti-SMA-cy3 (Sigma, C6198, 1:100), rat anti-PdgfrB (eBioscience, 14-1402-82, 1:100), rat anti-Ter-119 (R and D, MAB1125, 1:100), rabbit anti-Vegfr3 (ReliaTech, 102-PA22S, 1:50), rabbit anti-Collagen Type I (Millipore, AB765P, 1:100), goat anti-Collagen Type IV (Millipore, AB769, 1:100) and rabbit anti-ERG (Abcam ab110639, 1:100).

Techniques: Transfection, Quantitation Assay, Expressing, Mutagenesis, Fluorescence