Mouse EphB4 Antibody

EphB4 in Mouse Embryo.

EphB4 was detected in immersion fixed frozen sections of mouse embryo (15 d.p.c.) using Goat Anti-Mouse EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF446) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of Mouse EphB4 by Western Blot

Reduced Eph-B4 activity increases venous neointimal thickening. (A) Representative photomicrographs (left panel) and bar graph (right panel) showing AVF venous limb wall thickness in control and Eph-B4 het mice (day 21); *P = 0.047 (t-test). n = 8. Scale bar 25 µm. (B) Line graph showing infrarenal IVC diameter in control or Eph-B4 het mice; *P = 0.59 (ANOVA). n = 8–9. (C) Representative Western blot showing inhibited tyrosine phosphorylation in the Y774F-Eph-B4 mutant compared to the WT-Eph-B4 construct (0–60 min). (D) Bar graph showing Ephrin-B2/Fc stimulated COS cell migration after transfection with WT-Eph-B4 or Y774F-Eph-B4 plasmids. P < 0.0001 (ANOVA); *P < 0.0001 Ephrin-B2/Fc WT-Eph-B4 vs Y774F-Eph-B4. n = 3–4. (E) Representative photomicrographs (left panel) showing AVF venous wall (elastin stain) in control mice or mice treated with WT-Eph-B4 or mutant Y774F-Eph-B4. Arrow heads denote neointimal thickness. Scale bar, 25 µm. Bar graph (right panel) showing quantification of AVF venous wall thickness in control mice (white bar) or mice treated with WT-Eph-B4 (gray bar) or mutant Y774F-Eph-B4 (blue bar), day 21; P = 0.035 (ANOVA). *P = 0.038 (WT-Eph-B4 vs Y774F-Eph-B4; post hoc). n = 5–7. (F) Line graph showing infrarenal IVC diameter in mice with AVF treated with WT-Eph-B4 (gray line) or mutant Y774F-Eph-B4 (purple line) compared to control (black line); *P = 0.005 (ANOVA). n = 5–11. Data represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29133876), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human EphB4 by Western Blot

Increased Eph-B4 and Ephrin-B2 expression during adaptive venous remodeling. (A) Western blot and adjacent bar graph of densitometry showing human Eph-B4 expression in AVF venous limb compared to normal vein. *P = 0.0016; t-test. n = 3–4. (B) Line graphs show expression of Eph-B4 (blue) and Ephrin-B2 (red) in the AVF venous limb compared to sham IVC; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0123, Eph-B4; P = 0.0041, Ephrin-B2; post hoc); **P < 0.05 (P < 0.0001, Ephrin-B2; post hoc). n = 5–8. (C) Western blots showing Eph-B4 and Ephrin-B2 protein expression in AVF venous limb compared to sham IVC. n = 3–5. (D) Graphs showing densitometry of Eph-B4 (left panel) and Ephrin-B2 (right panel) expression in the AVF venous limb compared to sham IVC; *P < 0.05 (P < 0.0001, Eph-B4 day 7, AVF vs sham; P < 0.0001, Eph-B4 day 21, AVF vs sham; P < 0.0001, Ephrin-B2 day 7, AVF vs sham; post hoc). n = 3–5. (E) Diagram of rat model showing location of infrarenal IVC pericardial patch exposed to an aortocaval AVF (n = 6 per group). (F) Representative Western blot (upper panel) showing Eph-B4 and Ephrin-B2 expression in patch neointima (day 14) of control vein compared to patch neointima of AVF vein. Graphs (lower panel) show quantification of western blot bands; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0003, Eph-B4; P = 0.0043, Ephrin-B2; post hoc). n = 3. (G) Representative photomicrographs (upper panel) showing Eph-B4 (green) and Ephrin-B2 (red) immunoreactive signal (day 14). White arrowheads indicate colocalization of Eph-B4 and Ephrin-B2. L, vessel lumen. Graph (lower panel) shows quantification of immunoreactive signal; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0136 Eph-B4; P < 0.0001 Ephrin-B2; post hoc). n = 3. Scale bar 100 µm. Data represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29133876), licensed under a CC-BY license. Not internally tested by R&D Systems.