Journal: The Journal of Neuroscience

Article Title: EphB3: An Endogenous Mediator of Adult Axonal Plasticity and Regrowth after CNS Injury

doi: 10.1523/JNEUROSCI.4797-05.2006

Figure Lengend Snippet: Expression of EphB3 protein in the injured adult optic nerve and macrophages. A, Immunoblots of protein preparations from uninjured (UI) and injured adult optic nerves 8 d after injury (8) probed using an anti-EphB3 antibody. A signal at ∼110 kDa corresponding to the expected size for EphB3 protein was detected in both samples. This protein band was not present in optic nerve tissue obtained from EphB3 null animals (KO). GAPDH-immunopositive bands used as protein loading controls are shown below. B, ED1-positive macrophages isolated from segments of injured adult optic nerve in culture. C, The macrophages shown in B bound exogenously applied EphrinB3-Fc protein resulting in a punctate–aggregate pattern of cell-surface labeling. D, ED1-positive macrophages from segments of injured adult optic nerve in culture derived from an EphB3 homozygous null animal. E, Exogenously applied EphrinB3-Fc protein failed to bind to the EphB3 null macrophages shown in D. F, ED1-positive macrophages in an adult optic nerve 5 d after injury (green). G, EphrinB3-Fc binding in the same region shown in D (red). H, Colocalization of ED1 immunoreactivity with EphrinB3-Fc binding. Scale bars: (in B) B–E, 20 μm; (in F) F–H, 10 μm.

Article Snippet: After transfer to nitrocellulose, blots were blocked with PBS/5% anti-donkey serum or dry milk and probed with anti-mouse EphB3 (1:1000; R & D Systems) or anti-human EphrinB3 (1:500; R & D Systems).

Techniques: Expressing, Western Blot, Isolation, Labeling, Derivative Assay, Binding Assay

Journal: The Journal of Neuroscience

Article Title: EphB3: An Endogenous Mediator of Adult Axonal Plasticity and Regrowth after CNS Injury

doi: 10.1523/JNEUROSCI.4797-05.2006

Figure Lengend Snippet: EphrinB3 mRNA and protein expression in the adult retina and on RGC axons. A, EphrinB3 mRNA expression in the ganglion cell layer and the inner nuclear layer of the retina from a normal adult mouse. [See D for location of ganglion cell layer (GCL) and inner nuclear layer (INL).] B, EphrinB3 mRNA expression in the ganglion cell layer and the inner nuclear layer 4 d after optic nerve injury. C, EphrinB3 mRNA expression in the ganglion cell layer and the inner nuclear layer 12 d after optic nerve injury. D, Retinal section from a normal adult mouse exposed to the EphrinB3 sense control probe. E, EphrinB3 mRNA expression in the optic nerve of a normal adult mouse. A faint hybridization signal is detected in cells distributed in a row-like manner. F, EphrinB3 mRNA expression in the optic nerve of an adult mouse 4 d after optic nerve injury. G, EphrinB3 mRNA expression in the optic nerve of an adult mouse 12 d after optic nerve injury. H, Anti-EphrinB3 immunoblot. A faint signal is detected in the protein sample from uninjured optic nerves (UI). A band at ∼65 kDa, the expected size of EphrinB3, is present in the optic nerve samples 8 d after injury (8). I, Uninjured optic nerve section after staining using anti-EphrinB3 antibody. There is diffuse staining particularly at the ONH region. J, Optic nerve (ON) tissue section 12 d after injury stained with anti-EphrinB3 antibody. A strong signal was detected in the optic nerve proximal to the injury site. CT, Connective tissue; L, lesion site. K, Section from the same optic nerve shown in J after staining with a secondary antibody alone. L, Recombinant EphB3-Fc protein injected into the adult optic nerve binds to RGC axons. EphB3-Fc was detected using an anti-Fc antibody. M, Fc control protein injected into the adult optic nerve is distributed as diffuse punctate aggregates and does not result in apparent RGC axon binding. Scale bars: A–G, I–K, 100 μm; L, M, 20 μm.

Article Snippet: After transfer to nitrocellulose, blots were blocked with PBS/5% anti-donkey serum or dry milk and probed with anti-mouse EphB3 (1:1000; R & D Systems) or anti-human EphrinB3 (1:500; R & D Systems).

Techniques: Expressing, Control, Hybridization, Western Blot, Staining, Recombinant, Injection, Binding Assay

Journal: The Journal of Neuroscience

Article Title: EphB3: An Endogenous Mediator of Adult Axonal Plasticity and Regrowth after CNS Injury

doi: 10.1523/JNEUROSCI.4797-05.2006

Figure Lengend Snippet: EphB3-Fc protein supports adult RGC axon outgrowth. A, Adult retinal explant grown on a substratum coated with low levels of laminin (2.5 μg/ml). This level of laminin coating supports only minimal axon outgrowth (arrow points to an axon). B, Adult retinal explant grown for 3 d on a substratum coated with low laminin (2.5 μg/ml) and EphB3-Fc (9 μg/ml). The presence of EphB3-Fc increased the amount of axon outgrowth from adult retinal explants. C, Growth cone extending on low laminin (2.5 μg/ml) and EphB3-Fc (9 μg/ml). D, Growth cone extending on L1 (0.25 μg/ml) and EphB3-Fc (10 μg/ml). E, Anti-tubulin staining in a growth cone similar to C. F, Phalloidin staining in the same growth cone as in E. G, Anti-GAP-43 immunostaining in a retinal axon grown on a laminin substratum (10 μg/ml). H, Immunostaining showing L1 protein expression on RGC axons within the optic nerve 12 d after injury. The asterisk indicates the injury site. I, Graph showing the dose–response relationship between the mean axon number with increasing concentrations of EphB3-Fc protein used for coating onto a laminin substratum. The numbers at the top of each column indicate the number of explants examined for the given experimental condition. J, Graph showing the relationship between total axon length (mean ± SEM) with increasing concentrations of EphB3-Fc protein used for substratum coating. The numbers at the top of each column indicate the number of explants examined for the given experimental condition. K, Graph showing the dose–response relationship between the percentage of retinal explants with axon outgrowth and increasing amounts of EphB3-Fc protein used for coating onto an L1 substratum. The numbers in parentheses represent the numbers of explants used in each condition. Data from five independent experiments (mean ± SEM) are shown. The percentage of retinal explants from EphrinB3 null animals with axon outgrowth on an L1/EphB3-Fc substratum is indicated by the open bar. Data from three independent experiments (mean ± SEM) are shown. L, Graph showing the dose–response relationship between the amount of outgrowth with increasing amounts of EphB3-Fc protein used for coating onto a, L1 substratum. The numbers in parentheses represent the total number of explants used in each condition. Data from three independent experiments (mean ± SEM) are shown. The amount of outgrowth from retinal explants of EphrinB3 null animals on an L1/EphB3-Fc substratum is indicated by the open bar. Data from three independent experiments (mean ± SEM) are shown. Scale bars: A, B, 50 μm; C, D, G, 5 μm; E, F, 2 μm; H, 100 μm. LN, Laminin.

Article Snippet: After transfer to nitrocellulose, blots were blocked with PBS/5% anti-donkey serum or dry milk and probed with anti-mouse EphB3 (1:1000; R & D Systems) or anti-human EphrinB3 (1:500; R & D Systems).

Techniques: Staining, Immunostaining, Expressing

Journal: bioRxiv

Article Title: Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling

doi: 10.1101/498261

Figure Lengend Snippet: ( A ) Quantification of the number of adhered and polarized SC seeded on substrate coated with EphrinB3 with or without FN. Data are expressed as ratio respect to Fc-coated surfaces. EphrinB3 (10µg/mL), EphrinB3 (10µg/mL)+FN (2µg/cm 2 ), n=7 per group. ( B 1 ) Diagram of the adhesion assay on FN. Coverslips divided by silicon strips were coated with Fc+FN (control) or EphrinB3+FN in each half. After strip removal, cells pre-incubated with IgG or anti-Integrinβ1 were seeded homogenously on the coverslip. ( B 2 ) Blocking Integrinβ1 decreased SC adhesion and polarization on EphrinB3+FN compared with Fc+FN, scale, bar 200µm. ( C ) Quantification of SC adhesion and polarization on EphrinB3 when SC are pre-incubated with IgG or anti-integrinß1, n=15-17 per group. ( D-E ) Western blot analysis shows increased expression of Integrinβ1 when SC are activated by myelin extracts. One-way ANOVA p=0.011, F(2,12)=6.62, followed by Tukey’s multiple comparisons test p<0,05, n=5 per group. In A, C */** are used for comparison of a group with its hypothetical mean: 1 by one sample two-tailed t-test, and #/## for comparison between two different groups by two-tailed Mann Whitney test.*/# means p<0.05; **/## means p<0.001.

Article Snippet: After electrophoresis, proteins were transferred electrophoretically to polyvinylidene difluoride membranes and probed with the following antibodies:anti-EphB6 (1:500, SAB4503476, Sigma), anti-EphB1 (1:500, SAB4500776, Sigma), anti-EphA4 (4µg/mL, 37-1600, ThermoFisher), anti-p-EphB1+2 (1:300, ab61791, Abcam), anti-p-EphA4 (1:300, EP2731, ECM Biosciences), anti-Integrinβ1 (1:500, 550531, BD Pharmingen), anti-EphrinB3 (1:250, 1µg/mL, AF395 R&D systems), anti-MBP (1:1000, ab980, Millipore), anti-GAPDH (1:5000, MAB374, Millipore) and anti-Actin (1:50000, A2228, Sigma).

Techniques: Cell Adhesion Assay, Stripping Membranes, Incubation, Blocking Assay, Western Blot, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling

doi: 10.1101/498261

Figure Lengend Snippet: ( A ) SC encountering CNS white matter ( B ) are activated by the myelin-associated EphrinB3 through EphB6-and EphA4-SC receptors. ( C, D ) The activation by phosphorylation of these receptors impairs SC adhesion to white matter and increases SC expression of Integrinβ1 promoting their adhesion to BV extracellular matrix ( E ). Lesions of white matter undergo the formation and/or remodeling of BV which increase expression of ECM adhesion molecules, such as FN, and further facilitate SC mobilization towards the lesion.

Article Snippet: After electrophoresis, proteins were transferred electrophoretically to polyvinylidene difluoride membranes and probed with the following antibodies:anti-EphB6 (1:500, SAB4503476, Sigma), anti-EphB1 (1:500, SAB4500776, Sigma), anti-EphA4 (4µg/mL, 37-1600, ThermoFisher), anti-p-EphB1+2 (1:300, ab61791, Abcam), anti-p-EphA4 (1:300, EP2731, ECM Biosciences), anti-Integrinβ1 (1:500, 550531, BD Pharmingen), anti-EphrinB3 (1:250, 1µg/mL, AF395 R&D systems), anti-MBP (1:1000, ab980, Millipore), anti-GAPDH (1:5000, MAB374, Millipore) and anti-Actin (1:50000, A2228, Sigma).

Techniques: Activation Assay, Expressing

Journal: Nature Communications

Article Title: EphB4 forward signalling regulates lymphatic valve development

doi: 10.1038/ncomms7625

Figure Lengend Snippet: ( a ) Biotinylated α-EphB4 selectively binds to EphB4-Fc, but not other EphB proteins (left); and biotinylated α-ephrinB2 selectively binds to ephrinB2-Fc but not to other ephrinB proteins (right). ( b ) Blocking activity of α-ephrinB2 measured by western blot (WB) analysis of EphB4 phosphorylation in HUVECs stimulated by overlaid ephrinB2-expressing 3T3 cells. ( c ) Agonistic activity of α-EphB4 measured by WB (left) and ELISA (right) of EphB4 phosphorylation in EphB4-expressing 3T3 cells treated with ephrinB2-Fc or α-EphB4. Dotted line indicates a cropped lane (full WB data in ). ( d ) Antagonistic activity of α-EphB4 Fab measured by ELISA of EphB4 phosphorylation in EphB4-expressing 3T3 cells. ( a , c , d ) Error bars, s.d. of technical triplicates. Ctrl, control; IP, immunoprecipitation.

Article Snippet: An identical procedure was used to determine the selectivity of anti-ephrinB2 except that the recombinant proteins of the extra cellular domains of ephrinB1, ephrinB2 and ephrinB3 (R&D system, 5 μg ml −1 ), and biotinylated anti-ephrinB2 (Genentech, 0.1 μg ml −1 ) were used.

Techniques: Blocking Assay, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: EphB4 forward signalling regulates lymphatic valve development

doi: 10.1038/ncomms7625

Figure Lengend Snippet: Treatment was started at P1 and mice were examined on P7. Scale bar, 1 mm. ( a ) Chylothorax in α-ephrinB2-treated mice. Exterior ( b ) and interior ( c ) views of the leg skins following injection of FITC-dextran into the hindlimb footpad. The main lymphatic collecting vessels are marked (arrowheads). Anti-ephrinB2 (α-ephrinB2)-treated animals exhibited abnormal outflow of FITC-dextran from collecting vessels to the pre-collector vessel branches. ( d ) Collecting vessels running between the lumbar (LLN) lymph nodes and renal lymph nodes (RLN). In control (Ctrl) mice, only a pair of collecting lymphatic vessel trunks was highlighted by FITC-dextran (left). In anti -ephrinB2-treated animals (right), FITC-dextran visibly diffused into the surrounding lymphatic network.

Article Snippet: An identical procedure was used to determine the selectivity of anti-ephrinB2 except that the recombinant proteins of the extra cellular domains of ephrinB1, ephrinB2 and ephrinB3 (R&D system, 5 μg ml −1 ), and biotinylated anti-ephrinB2 (Genentech, 0.1 μg ml −1 ) were used.

Techniques: Injection

Journal: Nature Communications

Article Title: EphB4 forward signalling regulates lymphatic valve development

doi: 10.1038/ncomms7625

Figure Lengend Snippet: Visualization of lymphatic vessels and valves (arrows, not all valves are marked) by footpad injection of FITC-lectin following antibody administration from P1. ( a ) Defective lymphatic valves with ring-like appearance (arrow heads) in P5 leg skins of mice treated with function-blocking anti-ephrinB2 (α-ephrinB2) antibody. Right panel, quantification of lymphatic valves. ( b ) Higher magnification view of defective lymphatic valves in P5 leg skins of mice treated with function-blocking α-ephrinB2 antibody. ( c ) Agonistic anti-EphB4 (α-EphB4) reverses lymphatic valve defects caused by function-blocking α-ephrinB2. P6 leg skins of neonatal mice are shown. Lower panel, quantification of lymphatic valves. ( d ) Abnormal lymphatic valves with ring-like appearance (arrow heads) in P7 leg skins of mice treated with antagonistic anti-EphB4 Fab (α-EphB4 Fab). Lower panel, quantification of lymphatic valves. Scale bars: ( a ), ( c ) and ( d ), 500 μm; ( b ), 100 μm. **** P < 0.0001, *** P < 0.001, ** P < 0.01 (two-tailed, unpaired Student's t -test), n =3 per treatment group (error bars, s.d.). NS, not significant.

Article Snippet: An identical procedure was used to determine the selectivity of anti-ephrinB2 except that the recombinant proteins of the extra cellular domains of ephrinB1, ephrinB2 and ephrinB3 (R&D system, 5 μg ml −1 ), and biotinylated anti-ephrinB2 (Genentech, 0.1 μg ml −1 ) were used.

Techniques: Injection, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: EphB4 forward signalling regulates lymphatic valve development

doi: 10.1038/ncomms7625

Figure Lengend Snippet: ( a ) Schematic representation of ephrinB2 mutants. ( b , d , e ) Visualization of mesenteric lymphatic vessels and valves (arrows) by immuno-staining for Prox-1. Strong α-smooth muscle actin (αSMA) staining highlights blood vessels. Scale bar, 500 μm. ( b ) Defective mesenteric lymphatic valve development in E18 ephrinB2 6YFΔV/6YFΔV embryos. ( c ) Compromised EphB4 activation in ephrinB2 6YFΔV/6YFΔV embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 (pEphB4) and total-EphB4 immunoblotting analysis. Ratios of pEphB4:EphB4 are graphed (right panel). ( d ) In utero treatment with agonist anti-EphB4 (α-EphB4) restores lymphatic valves in the mesentery of P0 ephrinB2 6YFΔV/6YFΔV mice. Right panel, quantification of mesenteric lymphatic valves, n =3 per genotype. ( e ) Normal mesenteric lymphatic valve development in ephrinB2 lac Z/6YFΔV neonatal mice (P3). Right panel, quantification of mesenteric lymphatic valves, n =3 per genotype. Statistical analysis in ( c – e ), two-tailed, unpaired Student's t -test, error bars, s.d. NS, not significant.

Article Snippet: An identical procedure was used to determine the selectivity of anti-ephrinB2 except that the recombinant proteins of the extra cellular domains of ephrinB1, ephrinB2 and ephrinB3 (R&D system, 5 μg ml −1 ), and biotinylated anti-ephrinB2 (Genentech, 0.1 μg ml −1 ) were used.

Techniques: Immunostaining, Staining, Activation Assay, Western Blot, In Utero, Two Tailed Test

Journal: Nature Communications

Article Title: EphB4 forward signalling regulates lymphatic valve development

doi: 10.1038/ncomms7625

Figure Lengend Snippet: ( a ) Visualization of mesenteric lymphatic vessels and valves by immunostaining for Prox-1 in E18 embryos. Scale bar, 500 μm. Right panel, quantification of lymphatic valves, two-tailed, unpaired student's t -test, n =3 per genotype (error bars, s.d.). ( b ) Elevated EphB4 phosphorylaiton in ephrinB2 lac Z/ lacZ embryos. Total tissue lysates from E12.5 embryos were subjected to phospho-EphB4 and total EphB4 immunoblotting analysis. Ratios of pEphB4:total EphB4 are graphed. ** P <0.01 (two-tailed, unpaired student's t -test), n =6 for ephrinB2 +/ + , n =4 for ephrinB2 lac Z/ lacZ , error bars, s.d. NS, not significant.

Article Snippet: An identical procedure was used to determine the selectivity of anti-ephrinB2 except that the recombinant proteins of the extra cellular domains of ephrinB1, ephrinB2 and ephrinB3 (R&D system, 5 μg ml −1 ), and biotinylated anti-ephrinB2 (Genentech, 0.1 μg ml −1 ) were used.

Techniques: Immunostaining, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: EphB4 forward signalling regulates lymphatic valve development

doi: 10.1038/ncomms7625

Figure Lengend Snippet: ( a – c ) Visualization of mesenteric lymphatic vessels (L) and valves (arrows) by immunostaining for Prox-1 following treatments starting from P2. Blood vessels are highlighted by strong α-smooth muscle actin (αSMA) staining. Scale bar, 200 μm. ( a ) Agonistic anti-EphB4 (α-EphB4) reverses lymphatic valve defects caused by function-blocking anti-ephrinB2 (α-ephrinB2) in EphB4 ASKA ( EphB4 T693A/T693A ) neonatal mice. P7 mesenteric vessels are shown. Right panel, quantification of lymphatic valves. **** P <0.0001 (two-tailed, unpaired Student's t -test), n =3 per treatment group (error bars, s.d.). ( b ) NaPP1 treatment results in lymphatic valve defect in EphB4 T693A/T693A neonatal mice. P4 mesenteric vessels are shown. Right panel, quantification of lymphatic valves. **** P <0.0001(two-tailed, unpaired Student's t -test), n =3 per treatment group (error bars, s.d.). ( c ) NaPP1 treatment has no effect on lymphatic valves in wild-type neonatal mice. P4 mesenteric vessels are shown. Right panel, quantification of lymphatic valves, n =3 per treatment group, two-tailed, unpaired Student's t -test, error bars, s.d. ( d ) Schematic representation of EphB4 ASKA mutant (T693A). EphB4 ASKA is susceptible to inhibition by NaPP1. Ctrl, control; NS, not significant.

Article Snippet: An identical procedure was used to determine the selectivity of anti-ephrinB2 except that the recombinant proteins of the extra cellular domains of ephrinB1, ephrinB2 and ephrinB3 (R&D system, 5 μg ml −1 ), and biotinylated anti-ephrinB2 (Genentech, 0.1 μg ml −1 ) were used.

Techniques: Immunostaining, Staining, Blocking Assay, Two Tailed Test, Mutagenesis, Inhibition