To stimulate EphB4 signaling in vivo, mice received 1 mg/kg of mouse ephrinB2‐Fc (R&D Systems) or control Fc (Abcam, Cambridge, UK) by intraperitoneal injections twice weekly, starting 3 days before the myoblast injection, according to published protocols 62 .
Mouse ephrinb2 fc
Manufactured by R&D Systems
Sourced in United Kingdom
Mouse ephrinB2-Fc is a recombinant fusion protein consisting of the extracellular domain of mouse ephrinB2 and the Fc region of human IgG1. It is used as a research tool in studies involving cell-cell interactions and signaling processes mediated by ephrinB2.
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Lab products found in correlation
5 protocols using mouse ephrinb2 fc
1
Stimulating EphB4 Signaling in Mice
2
Homo- and Heterodimerizing Agents Protocol
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Homo- and heterodimerizing agents AP20187 (Clackson et al., 1998 (link)), AP1887 (Yang et al., 2003 (link)), and AP21967 were obtained from Ariad Pharmaceuticals. Before stimulation, dimerizers were diluted in starving medium used for stimulation to the concentration indicated in the figures. Human IgG Fc fragment (Jackson ImmunoResearch Laboratories, Inc.), mouse ephrinB2-Fc, human ephrinB3-Fc, and human ephrinA5-Fc fusion proteins (R&D Systems) were used for stimulations. For preclustering Fc fragment and ephrin–Fc, fusion proteins were incubated with goat anti–human Fc at a ratio of 5:1 for 30 min at RT.
3
HUVEC Phospho-EphB4 Quantification
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Human umbilical vein endothelial cells (HUVEC) were seeded at the density of 500,000 cells/T25 flask and cultured overnight in M199 medium supplemented with 20% FBS, 100 μg/ml endothelial cell growth supplement (Sigma‐Aldrich, St. Louis, USA), 50 U/ml sodium heparin (Sigma‐Aldrich), and 1% penicillin/streptomycin. Afterward, cells were incubated with conditioned medium from either sEphB4 or control CD4 myoblasts that was previously kept on ice for 30 min with 2 μg/ml mouse ephrinB2‐Fc (R&D Systems) pre‐clustered with anti‐Fc Ab 16 and then warmed at 37°C for 15 min. After 30 min, HUVEC were lysed and the amount of phospho‐EphB4 was quantified using a human phosphotyrosine EphB4 ELISA kit (Raybiotech, Norcross, GA, USA) according to manufacturer's instructions.
4
Ephrin-B2 Stimulation Assay
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Following starvation, cells were stimulated with either mouse Ephrin-B2/Fc (2 µg/ml) or mouse IgG/Fc (2 µg/ml; R&D Systems) in PBS, for the designated amount of time, after which either the cell lysates or the conditioned media were collected.
5
Endothelial Cell Shear Stress Assay
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EC were cultured to confluence on collagen I-coated glass plates (StreamerTM Culture Slips, Flexcell Corporation). Confluent EC were serum-starved for 24 hr and then pre-treated with NVP-BHG712 (1 µM; Sigma Aldrich, St. Louis, MO) for 1 hr or stimulated with 2 µg/ml mouse Ephrin-B2/Fc (R&D Systems) for 1 hr and compared to control. After pre-treatment (37 °C, 5% CO2), cells were exposed to steady laminar flow in a parallel-plate flow chamber with circulating EBM-2 medium (0% FBS, 37 ± 0.5 °C, 1 hr). Wall shear stress was calculated by the formula τ = 6 μQ/bh2, where μ is the viscosity of the fluid, Q is the flow volume (ml/s), b is the width of the flow channel in cm, and h is the height of the flow channel in cm; shear stress was set at 0 dynes/cm2 or 20 dynes/cm2. After shear stress treatment, the EC on the slides were removed with RIPA lysis buffer and analyzed.
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