Journal: The Journal of Biological Chemistry

Article Title: Identification and characterization of Nanobodies targeting the EphA4 receptor

doi: 10.1074/jbc.M116.774141

Figure Lengend Snippet: Nb 39 and Nb 53 do not completely inhibit ephrin-A5 binding to EphA7. With Alphascreen technology we determined whether Nb 39 and Nb 53 could inhibit ephrin-A5 binding to EphA7. These Nbs did not completely inhibit the interaction between ephrin-A5 and EphA7 at the highest concentrations tested. This assay was done in triplicate, and data are represented as the mean ± S.D.

Article Snippet: To test the inhibition of EphA7 and ephrin-A5 interaction, His-tagged human ephrin-A5 (Sino Biological, Beijing, China) was biotinylated with a five-times molar excess of EZ link NHS biotin (Thermo Fisher Scientific) as is described for the Nbs.

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: Soft matter

Article Title: N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

doi: 10.1039/c7sm01214d

Figure Lengend Snippet: Fig. 1 Reconstitution of recombinant Fc-chimera synaptic adhesion pro- teins in a fluid lipid bilayer (POPC). (A) Schematic of the reconstitution of ephrinA5 and N-cadherin into small unilamellar vesicles (SUVs) and (B) formation of lipid bilayer (POPC) after sonication and extrusion (100 nm polycarbonate membrane).

Article Snippet: EphrinA5 (recombinant Human ephrinA5 Fc-Chimera, CF), N-cadherin (recombinant Human N-cadherin Fc-Chimera, CF), the primary antibody goat anti-human ephrinA5 (incorporated ephrinA5 receptor), and the primary antibody goat anti-human N-cadherin (incorporated N-cadherin ligand) were purchased from R&D Systems, whereas the mouse anti-rat ephrinA5 antibody (ligand), mouse anti-N-cadherin, mouse anti-Synaptophysin (7H12) and rabbit anti-Tubulin beta 3 (TUJ1; D71G9) were purchased from Cell Signaling Technology.

Techniques: Recombinant, Sonication, Membrane

Journal: Soft matter

Article Title: N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

doi: 10.1039/c7sm01214d

Figure Lengend Snippet: Fig. 3 Neuronal adhesion on modified lipid bilayer and PDL + laminin coated substrates. (A) Overview of neuronal adhesion on PDL + laminin (i, ii and ii0), N-cadherin-doped bilayer (iii, iv and iv0), and ephrinA5-doped lipid bilayer (v, vi and vi0) (red arrows indicate degenerated neurites) at DIV7 and DIV14. Neurons on PDL + laminin (i and ii) show strong cell adhesion to the surface at DIV7 and DIV14, adhesion on the N-cadherin bilayer at DIV7 and DIV14 shows homogeneous distribution of neuronal cells while adhesion on ephrinA5 bilayers leads to cell aggregates both at DIV7 and DIV14. (B) Determination of neurite length of cortical neurons on PDL + laminin coated surfaces and N-cadherin and ephrinA5 lipid bilayer at DIV1. (C) Quantification of neuronal cell growth and number of live cells at DIV7 and DIV14 on various substrates.

Article Snippet: EphrinA5 (recombinant Human ephrinA5 Fc-Chimera, CF), N-cadherin (recombinant Human N-cadherin Fc-Chimera, CF), the primary antibody goat anti-human ephrinA5 (incorporated ephrinA5 receptor), and the primary antibody goat anti-human N-cadherin (incorporated N-cadherin ligand) were purchased from R&D Systems, whereas the mouse anti-rat ephrinA5 antibody (ligand), mouse anti-N-cadherin, mouse anti-Synaptophysin (7H12) and rabbit anti-Tubulin beta 3 (TUJ1; D71G9) were purchased from Cell Signaling Technology.

Techniques: Modification

Journal: Soft matter

Article Title: N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

doi: 10.1039/c7sm01214d

Figure Lengend Snippet: Fig. 4 Co-localization of bilayer incorporated and native protein during cortical neuron adhesion on a SLB (DIV14). (A) Interaction of ephrinA5-Fc (red, DyLight594) and EphrA receptor (mouse anti-rat EphrA5, blue, AlexaFluor 350) along the axons and dendrites of cortical neurons. (B) Homophilic interaction of N-cadherin proteins. Co-localization (yellow) of membranous N-cadherin (green, AlexaFluor 488) and N-cadherin-Fc (red, DyLight 594). Scale bar 50 mm.

Article Snippet: EphrinA5 (recombinant Human ephrinA5 Fc-Chimera, CF), N-cadherin (recombinant Human N-cadherin Fc-Chimera, CF), the primary antibody goat anti-human ephrinA5 (incorporated ephrinA5 receptor), and the primary antibody goat anti-human N-cadherin (incorporated N-cadherin ligand) were purchased from R&D Systems, whereas the mouse anti-rat ephrinA5 antibody (ligand), mouse anti-N-cadherin, mouse anti-Synaptophysin (7H12) and rabbit anti-Tubulin beta 3 (TUJ1; D71G9) were purchased from Cell Signaling Technology.

Techniques:

Journal: Soft matter

Article Title: N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

doi: 10.1039/c7sm01214d

Figure Lengend Snippet: Fig. 5 Maturation-related targeting of synaptophysin in cortical neurons. Neurons are labeled against TUJ1 (white), and synaptophysin (green) in (i–vi0) at DIV7 and DIV14. Synaptophysin expression in neuronal cells is shown on (i) PDL + laminin coating, (iii) N-cadherin lipid bilayer, and (v) ephrinA5 lipid bilayer. Red arrows indicate the localization of SLB incorporated proteins. Scale bar represents 100 mm.

Article Snippet: EphrinA5 (recombinant Human ephrinA5 Fc-Chimera, CF), N-cadherin (recombinant Human N-cadherin Fc-Chimera, CF), the primary antibody goat anti-human ephrinA5 (incorporated ephrinA5 receptor), and the primary antibody goat anti-human N-cadherin (incorporated N-cadherin ligand) were purchased from R&D Systems, whereas the mouse anti-rat ephrinA5 antibody (ligand), mouse anti-N-cadherin, mouse anti-Synaptophysin (7H12) and rabbit anti-Tubulin beta 3 (TUJ1; D71G9) were purchased from Cell Signaling Technology.

Techniques: Labeling, Expressing

Journal: Soft matter

Article Title: N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

doi: 10.1039/c7sm01214d

Figure Lengend Snippet: Fig. 6 Adhesion mechanism of cortical neurons on modified lipid membranes. (A) Schematic representation of cell adhesion and maturation of neuronal cells via homophilic interaction of N-cadherin in the cell membrane with N-cadherin-Fc in the SLB and associated neurite formation. a-Catenin, b-catenin linked to tyrosine kinase; P: phosphorylated; (B) schematic illustration of cell adhesion via EphrA receptors to SLB-incorporated ephrinA5-Fc. Interactions of ephrinA5-Fc and EphrA receptor after endocytosis trigger changes in neurite outgrowth. E: endocytosis of the EphrA receptor–ligand complex. (C) Immunocytochemistry of co-expressed phosphorylated tyrosine kinases, (PY-99, red) in cortical neurons after DIV7 on N-cadherin and ephrinA5 doped lipid bilayers. Endogenously expressed EphrA receptor and N-cadherin are visualized in green. Scale bar 10 mm.

Article Snippet: EphrinA5 (recombinant Human ephrinA5 Fc-Chimera, CF), N-cadherin (recombinant Human N-cadherin Fc-Chimera, CF), the primary antibody goat anti-human ephrinA5 (incorporated ephrinA5 receptor), and the primary antibody goat anti-human N-cadherin (incorporated N-cadherin ligand) were purchased from R&D Systems, whereas the mouse anti-rat ephrinA5 antibody (ligand), mouse anti-N-cadherin, mouse anti-Synaptophysin (7H12) and rabbit anti-Tubulin beta 3 (TUJ1; D71G9) were purchased from Cell Signaling Technology.

Techniques: Modification, Membrane, Immunocytochemistry

Journal: Soft matter

Article Title: N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

doi: 10.1039/c7sm01214d

Figure Lengend Snippet: Fig. 7 Analysis of neuronal activity on artificial lipid membranes. Representative fluorescence traces of average network activity on different substrates. (A) Analysis of active cells on PDL-laminin substrates, N-cadherin, or ephrinA5 lipid membranes indicates that, in contrast to neurons on ephrinA5 membranes or PDL + laminin substrates, neurons on N-cadherin membranes already show a high neural activity at DIV7. (B) Relative change in fluorescence intensity of active cells on PDL + laminin substrates or artificial membranes with N-cadherin or ephrinA5 at DIV7 and DIV14. Calcium imaging indicates strong activity of neurons on N-cadherin lipid bilayers at DIV7.

Article Snippet: EphrinA5 (recombinant Human ephrinA5 Fc-Chimera, CF), N-cadherin (recombinant Human N-cadherin Fc-Chimera, CF), the primary antibody goat anti-human ephrinA5 (incorporated ephrinA5 receptor), and the primary antibody goat anti-human N-cadherin (incorporated N-cadherin ligand) were purchased from R&D Systems, whereas the mouse anti-rat ephrinA5 antibody (ligand), mouse anti-N-cadherin, mouse anti-Synaptophysin (7H12) and rabbit anti-Tubulin beta 3 (TUJ1; D71G9) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Fluorescence, Imaging

Journal: Cell Genomics

Article Title: Single-cell profiling of bone metastasis ecosystems from multiple cancer types reveals convergent and divergent mechanisms of bone colonization

doi: 10.1016/j.xgen.2025.100888

Figure Lengend Snippet: Bone stromal drives divergent bone colonization and immune evasion mechanisms (A) Analysis of cell-cell communication signal flow. Outgoing signal strength is shown on the x axis and incoming signal strength on the y axis, comparing the Mφ-OC and Treg-Tex archetypes with healthy samples serving as references. (B) Identification of key ligand-receptor pairs that differentially regulate the OC populations. This analysis compares the relative signaling strengths between the Mφ-OC and Treg-Tex archetypes, focusing on osteoclasts as the signal receivers (from A). (C) Schematic illustration of in vitro experimental validation for estimated signaling molecules. CD14 + monocytes isolated from human peripheral blood were enriched for osteoclastogenesis induction, with selected factors added to the culture medium to test their predicted roles in regulating differential osteoclastogenesis. Osteoclastogenesis was then evaluated by both qPCR and TRAP staining. (D) qPCR analysis of osteoclast signature genes to validate differential osteoclastogenesis regulation by estimated signaling molecules. Each signaling factor was tested using graded concentrations: TWEAK (TNFSF12; 0.1, 1, 10 ng/μL), COMP (5, 50, 500 ng/μL), and NRG1 (10, 100, 1000 ng/μL), TNFSF10 (1, 10, 100 ng/μL), SEMA4A (1, 10, 100 ng/μL), EFNA5 (1, 10, 100 ng/μL), BMP8A (1, 10, 100 ng/μL). Each condition has five replicates. Statistical significance was assessed using one-way ANOVA, with significance levels: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: recombinant human EFNA5 , Sino Biological , Cat#10192-H08H-100.

Techniques: In Vitro, Biomarker Discovery, Isolation, Staining