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: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Immunostaining against Doc2B (green) and EphB2 (red) in the mouse hippocampus region. Nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 500 μm. ( B ) Separation of pre- and postsynaptic densities from purified synaptosomes. Syn, synaptosome; Pre, presynaptic elements; Post, postsynaptic elements; Extra, extrajunctional synaptic elements. ( C and D ) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of mCherry-tagged Doc2B and Flag-tagged EphB2 in HEK293 cells. Cell lysates were immunoprecipitated by anti-mCherry antibody (C) or anti-Flag antibody (D), followed by immunoblotting with indicated antibody. ( E ) Schematic representation of EphB2 constructs. ( F and G ) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of various combinations of plasmids containing EphB2 FL, EphB2-∆SP, EphB2-∆KSP, and/or mCherry-tagged Doc2B (F), EphB2 WT, KR, and/or mCherry-tagged Doc2B (G) in HEK293 cells. ( H and I ) In vitro phosphorylation assay using sumo-tagged EphB2 and GST-tagged Doc2B fragments (H) and GST-tagged Doc2B Mid or Y36F (I) in the presence or absence of ATP and APase. Asterisks show bands of GST-Doc2B Mid, Mid-L, and FL, respectively. CBB, Coomassie Brilliant Blue staining. ( J and K ) Western blot analysis of Doc2B phosphorylation following cotransfection of mCherry-tagged Doc2B WT or Y36F and/or EphB2 (J) and EphB2 WT, KR, or YYEE and/or mCherry-tagged Doc2B (K) in HEK293 cells. ( L ) Detection of Doc2B phosphorylation in cultured mouse cortex neurons by application of Fc (control) or preclustered Ephrin-B3-Fc. ( M and N ) Western blot analysis of Doc2B phosphorylation in EphB2 +/+ or EphB2 −/− mouse brains (M) in Doc2A-deficient neurons infected with Doc2B KD or control virus (N). Tubulin used as the reference protein. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by Student’s t test (** P < 0.01; **** P < 0.0001).

Article Snippet: To detect the binding of EphrinBs and EphB2, EGFP-EphB2–transfected HEK293 cells were incubated with preclustered EphrinB-Fc (human) (1 μg/ml) for 15 min. To facilitate preclustering, EphrinB1-Fc, EphrinB2-Fc, or EphrinB3-Fc fragments (Sino Biological) were mixed with recombinant anti–IgG1-Fc rabbit Antibody (Sino Biological) in a 1:2 ratio and incubated on ice for 1 hour.

Techniques: Immunostaining, Labeling, Purification, Western Blot, Cotransfection, Immunoprecipitation, Construct, In Vitro, Phosphorylation Assay, Staining, Cell Culture, Infection, Virus

Journal: Cell Death & Disease

Article Title: EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury

doi: 10.1038/cddis.2015.262

Figure Lengend Snippet: EphB3 and ephrinB3 levels are altered after SCI. Western blot analysis for EphB3 and ephrinB3 at 1 ( a ), 3 ( b ) and 7 ( c ) day(s) post-injury (dpi) as compared with sham surgery. Significant reductions in EphB3 ( d ) and ephrinB3 ( e ) protein levels are observed at 1 dpi with a subsequent trends back to pre-injury levels of the EphB3 receptor, but not the ephrinB3 ligand by 7 dpi. Protein levels are normalized to β -actin controls. * P <0.05

Article Snippet: The primary antibodies anti-EphB3 (mouse monoclonal; Abcam; diluted 1 : 1000), anti-ephrinB3 (goat polyclonal; Santa Cruz Biotechnology Inc., Dallas, TX, USA; diluted 1 : 200), anti-MBP (mouse monoclonal, Abcam, diluted 1 : 100) and anti- β -actin (mouse monoclonal; Cell Signaling, Danvers, MA, USA; diluted 1 : 5000) were diluted in 5% milk in TBS-T for overnight incubation at 4 °C.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury

doi: 10.1038/cddis.2015.262

Figure Lengend Snippet: Significant improvements in hindlimb locomotor behavior are observed in EphB3 −/− as compared with WT mice by 7 dpi that is maintained for 7 weeks ( a ). Western blot analysis of MBP and β -actin in WT and EphB3 −/− sham and SCI mice at 7 dpi ( b ). Significant reductions in MBP levels were observed in WT, but not in EphB3 −/− SCI tissues, as compared with sham controls when normalized to β -actin ( c ). MBP levels in EphB3 −/− spinal cord were significantly higher than WT SCI mice. * P <0.05; ** P <0.01; # P <0.05. *Compared with WT sham; # Compared with WT SCI

Article Snippet: The primary antibodies anti-EphB3 (mouse monoclonal; Abcam; diluted 1 : 1000), anti-ephrinB3 (goat polyclonal; Santa Cruz Biotechnology Inc., Dallas, TX, USA; diluted 1 : 200), anti-MBP (mouse monoclonal, Abcam, diluted 1 : 100) and anti- β -actin (mouse monoclonal; Cell Signaling, Danvers, MA, USA; diluted 1 : 5000) were diluted in 5% milk in TBS-T for overnight incubation at 4 °C.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury

doi: 10.1038/cddis.2014.165

Figure Lengend Snippet: EphB3 and ephrinB3 protein levels are reduced in the cortex post CCI injury. Low-magnification images of Nissl-stained coronal sections at 3 days post sham ( a ) and CCI injury ( b ) showing selective cortical tissue loss with sparing of hippocampal structures. ( c and d ) X-gal staining in ephrinB3 lacZ mice after sham and CCI injury, respectively, demonstrates reduced staining in the cortex (CTX) and corpus callosum (C). ( e and f ) Western blot analysis for EphB3 and ephrinB3 expression in the cortex at 3 days ( e ) and 7 days ( f ) post injury. Significant reductions in EphB3 expression were observed at 3 days, as measured by densitometry normalized to β -actin control levels ( g ), whereas ephrinB3 expression was reduced, significantly, at 3 days and non-significantly at 7 days compared with sham-injured levels ( h ). ** P <0.01; * P <0.05 compared with sham injury. n =5 per group and time point

Article Snippet: Protein samples were resolved on 10% SDS-PAGE gels and blotted onto PVDF membranes that were blocked with 5% BSA or milk in TBST buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween) and incubated with primary antibodies against ephrinB3 (1 : 200, Invitrogen, Carlsbad, CA, USA), EphB3 (1 : 8000, Abcam), phospho-tyrosine (1 : 1000, Cell Signaling, Danvers, MA, USA) or β -actin (1 : 8000, Cell Signaling) diluted in TBST-3% BSA or milk overnight at 4 °C.

Techniques: Staining, Western Blot, Expressing, Control