Human EphB4 Antibody

Detection of Human EphB4 by Simple Western^TM.

Simple Western lane view shows lysates of HUVEC human umbilical vein endothelial cells, loaded at 0.2 mg/mL. A specific band was detected for EphB4 at approximately 127 kDa (as indicated) using 50 µg/mL of Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western Blot Shows Human EphB4 Specificity by Using Knockout Cell Line.

Western blot shows lysates of HEK293T human embryonic kidney parental cell line and EphB4 knockout HEK293T cell line (KO). PVDF membrane was probed with 2 µg/mL of Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for EphB4 at approximately 140 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Mouse EphB4 by Western Blot

EphB4 expression in transfected A375 melanoma cells and corresponding tumor xenografts. Western blot analysis of EphB4 and its ligand EphrinB2 in A375, A375-pIRES, and A375-EphB4 whole cell lysates (A) and tumor lysates (E). Anti-beta -actin served as loading control. (B) Relative mRNA expression of EphB4 and EphrinB2 in A375, A375-pIRES, and A375-EphB4 cells, analyzed by quantitative real-time RT-PCR was normalized to the constitutive expression level of beta-actin and to expression level in wild-type A375 melanoma cells using the delta delta CT method (2−∆∆ct) resulting in a value of 1 for A375 cells. Values represent mean ± SEM of at least three independent experiments each performed in triplicate (* p < 0.05, *** p < 0.001). (C) EphB4 phosphorylation was analyzed by pEphB4-ELISA in whole cell lysates of A375 EphB4 cells incubated with different concentrations of sEphrinB2-Fc (0; 0,5; 1 µg/mL) for 15, 30, and 60 min. Values represent mean ± SD from one of at least three independent experiments, each performed in duplicate. To rule out influence of sEphrinB2 on total EphB4 protein amount, EphB4 was analyzed in the same cell lysates by western blot analysis (figure shows one representative blot out of three independent experiments performed in duplicate) ranging from 92% to 112% of control as calculated after densitometric analysis. Anti beta-actin served as loading control. (D) Immunohistochemical detection of EphB4 in acetone-fixed cryosections of A375 pIRES and A375 EphB4 tumor xenografts using goat anti EphB4 antibody. Sections stained without the primary antibody served as negative control. Scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29462967), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse EphB4 by Western Blot

EphB4 expression in transfected A375 melanoma cells and corresponding tumor xenografts. Western blot analysis of EphB4 and its ligand EphrinB2 in A375, A375-pIRES, and A375-EphB4 whole cell lysates (A) and tumor lysates (E). Anti-beta -actin served as loading control. (B) Relative mRNA expression of EphB4 and EphrinB2 in A375, A375-pIRES, and A375-EphB4 cells, analyzed by quantitative real-time RT-PCR was normalized to the constitutive expression level of beta-actin and to expression level in wild-type A375 melanoma cells using the delta delta CT method (2−∆∆ct) resulting in a value of 1 for A375 cells. Values represent mean ± SEM of at least three independent experiments each performed in triplicate (* p < 0.05, *** p < 0.001). (C) EphB4 phosphorylation was analyzed by pEphB4-ELISA in whole cell lysates of A375 EphB4 cells incubated with different concentrations of sEphrinB2-Fc (0; 0,5; 1 µg/mL) for 15, 30, and 60 min. Values represent mean ± SD from one of at least three independent experiments, each performed in duplicate. To rule out influence of sEphrinB2 on total EphB4 protein amount, EphB4 was analyzed in the same cell lysates by western blot analysis (figure shows one representative blot out of three independent experiments performed in duplicate) ranging from 92% to 112% of control as calculated after densitometric analysis. Anti beta-actin served as loading control. (D) Immunohistochemical detection of EphB4 in acetone-fixed cryosections of A375 pIRES and A375 EphB4 tumor xenografts using goat anti EphB4 antibody. Sections stained without the primary antibody served as negative control. Scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29462967), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse EphB4 by Western Blot

EphB4 expression in transfected A375 melanoma cells and corresponding tumor xenografts. Western blot analysis of EphB4 and its ligand EphrinB2 in A375, A375-pIRES, and A375-EphB4 whole cell lysates (A) and tumor lysates (E). Anti-beta -actin served as loading control. (B) Relative mRNA expression of EphB4 and EphrinB2 in A375, A375-pIRES, and A375-EphB4 cells, analyzed by quantitative real-time RT-PCR was normalized to the constitutive expression level of beta-actin and to expression level in wild-type A375 melanoma cells using the delta delta CT method (2−∆∆ct) resulting in a value of 1 for A375 cells. Values represent mean ± SEM of at least three independent experiments each performed in triplicate (* p < 0.05, *** p < 0.001). (C) EphB4 phosphorylation was analyzed by pEphB4-ELISA in whole cell lysates of A375 EphB4 cells incubated with different concentrations of sEphrinB2-Fc (0; 0,5; 1 µg/mL) for 15, 30, and 60 min. Values represent mean ± SD from one of at least three independent experiments, each performed in duplicate. To rule out influence of sEphrinB2 on total EphB4 protein amount, EphB4 was analyzed in the same cell lysates by western blot analysis (figure shows one representative blot out of three independent experiments performed in duplicate) ranging from 92% to 112% of control as calculated after densitometric analysis. Anti beta-actin served as loading control. (D) Immunohistochemical detection of EphB4 in acetone-fixed cryosections of A375 pIRES and A375 EphB4 tumor xenografts using goat anti EphB4 antibody. Sections stained without the primary antibody served as negative control. Scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29462967), licensed under a CC-BY license. Not internally tested by R&D Systems.