ephb4 specific antibody  (Novus Biologicals)

Journal: Oncotarget

Article Title: Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

doi:

Figure Lengend Snippet: (A) The H200 antibody recognizes a 200 amino acid sequence in the extracellular domain of EphB4 spanning the cysteine rich domain (CYS) and the first fibronectin type III repeat (FIII). LBD—globular ligand binding domain, SAM—sterile alpha motif, PDZ—PDZ domain. (B) Flow cytometry comparing parental MCF10A cells with EphB4 over-expressing MCF10A-B4 cells using the H200 antibody showing an increase in fluorescence of the EphB4 over-expressing cells. (C) Western blot analysis with the H200 antibody identifying EphB4 over-expressed in the MCF10A-B4 cells (B4) compared with the parental empty vector only MCF10A-VO cells. β-actin was used to demonstrate equal loading. (D) Immunofluorescence using the H200 antibody to identify EphB4 (green) over-expressed in the MCF10A-B4 cells compared to the parental cells transfected with the empty vector (VO). Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Co-localisation of EphB4 and F-actin (yellow). Bar = 10 μm. (E) Photomicrographs of cells treated with 0.4 μg/ml H200 antibody added to confluent monolayers of SW480 colon cancer cells for different lengths of time. (F) Trypan blue assay of SW480 cells treated with four different EphB4-specific antibodies for 48 h. Antibodies included the Ziemiecki lab rabbit polyclonal antibody (S), EphB4 (N-19) (N), EphB4 (H-200) (H) and EphB4 (C-16) (C) all from Santa Cruz Biotechnology. The H200 antibody showed a reduction in the number of viable cells compared to the control untreated (Con) and the other three antibodies irrespective of the presence (FCS) or absence (CLM) of complement. (G) Trypan blue assay of SW480 cells treated with either 0.2 μg/ml or 1 μg/ml H200 for 48 or 72 h. A significant reduction in the number of viable cells was seen at 72 h with the low concentration ( p < 0.05) and at 48 h with the higher concentration ( p < 0.001), with no viable cells remaining after 72 h treatment with 1 μg/ml H200. (H) Caspase-3 assay of SW480 cells treated with different concentrations of H200 (as per Figure ). The reduction in cell viability correlates with an increase in caspase-3 activity, statistically significant at 72 h with 0.2 μg/ml ( p = 0.002) and at 48 h with 1 μg/ml ( p = 0.003).

Article Snippet: Three commercially available human EphB4-specific polyclonal antibodies EphB4 (N-19), EphB4 (H-200) and EphB4 (C-16) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Sequencing, Ligand Binding Assay, Sterility, Flow Cytometry, Expressing, Fluorescence, Western Blot, Plasmid Preparation, Immunofluorescence, Transfection, Control, Concentration Assay, Caspase-3 Assay, Activity Assay

Journal: Oncotarget

Article Title: Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

doi:

Figure Lengend Snippet: (A) The H200 pAb was tested using several different cell lines of colon (SW480, SW620), breast (MDA-MB-231, MCF-7, MCF10A), bladder (T24, HT119), endothelial (HUVEC), prostate (PC3, LNCaP) and osteosarcoma (BTK143, MG63) origins and after 48 h a significant reduction in the number of viable cells, determined using a trypan blue assay, when compared with untreated control cells was seen for all but T24, MCF10A, LNCaP, HUVEC and MG63. (B) Antibody targeting of EphB4 inhibits tumour cell colonisation of soft agar. MDA-MB-231, MDA-MB-468 and MCF10A cells were suspended in soft agar in the presence of EphB4 antibody (10 μg/ml) (Ab treated) or PBS vehicle control (Control) administered at the time of suspension. Colonies of more than 5 cells were counted from four different low power fields and the results are represented graphically. (C) Treatment with H200 prevented tubular nework formation on Matrigel. MDA-MB-468, MDA-MB-231, MCF-7 and MCF10A cells were plated on Matrigel in the presence of EphB4 antibody (10 μg/ml) (Ab treated) or PBS vehicle control (Control) and images representative of each assay were taken at 20× magnification after 4 days. (D) RT-PCR analysis of EphB4 gene expression (PCR product = 1187 bp) in breast cancer cell lines treated with antibody or untreated (control). Expression of the PBGD housekeeping gene (377 bp) was used as a control for the integrity of the cDNA and relative comparison of EphB4 expression levels. M1 = pUC19/ Hpa II marker, M2 = Lambda/ Hind III. (E) Western blot analysis showing that treatment with H200 causes loss of EphB4 protein at 72 h. β-actin was detected as a loading control and the total amount of EphB4 protein normalized to this using densitometry (values expressed as percentage of control). (F) Graph showing effect of H200 antibody on total EphB4 levels (relative to control) with a significant reduction seen at 72 h ( p = 0.0001).

Article Snippet: Three commercially available human EphB4-specific polyclonal antibodies EphB4 (N-19), EphB4 (H-200) and EphB4 (C-16) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Control, Suspension, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Comparison, Marker, Western Blot

Journal: Oncotarget

Article Title: Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

doi:

Figure Lengend Snippet: (A) Antibodies specifically recognizing Peptide 7 were isolated from the H200 polyclonal preparation. Western blot analysis shows that these antibodies (H7) specifically identify EphB4 with less non-specific cross-reactivity than the H200 antibody. β-actin was used as a loading control. (B) Western blot and graph showing that in a similar manner to H200, the H7 antibody, tested at two different dilutions, reduces total EphB4 protein level with a significant reduction seen at 72 h ( p = 0.0001). (C) Immunoprecipitation of cell lysates from MCF-7 breast cancer cells showing treatment with the H7 antibody causes phosphorylation of the EphB4 protein, seen at 5 min after addition and maintained at 30 min (yellow box). Cells were stimulated with soluble clustered ephrin-B2-Fc ligand (eB2) as a positive control for phosphorylation with clustered Fc fragment only (Fc) as the negative control for this. (D) Alignment of the EphB4 sequence of peptides 1 and 2, which includes peptide 7 sequence AGSCVVDA (red box) with the corresponding sequence from EphA3. Asterisks identify the amino acids identified as contributing to the third ligand binding domain. (E) Comparison of the peptide sequence used to raise EphB4 monoclonal antibodies (green) with other members of the human Eph family. Amino acids identical to the EphB4 sequence are shaded in grey, homologous amino acids are shaded pink. The antigenic aspartic acid reside is within a red box. (F) Comparison of the peptide sequence used to raise human EphB4 monoclonal antibodies (green) with EphB4 sequences from other species. Amino acids identical to the human EphB4 sequence are shaded in grey. (G) Western blot analysis using the H200 and 13A7 antibodies with recombinant human EphB4 extracellular domain (rhEphB4ecd) (H) and recombinant mouse EphB4 extracellular domain Fc fusion protein (rmEphB4ecd-Fc) (M). The arrows indicate the position of the proteins compared to the molecular weight marker for which sizes are indicated on the left.

Article Snippet: Three commercially available human EphB4-specific polyclonal antibodies EphB4 (N-19), EphB4 (H-200) and EphB4 (C-16) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Isolation, Western Blot, Control, Immunoprecipitation, Phospho-proteomics, Positive Control, Negative Control, Sequencing, Ligand Binding Assay, Comparison, Bioprocessing, Recombinant, Molecular Weight, Marker

Journal: Oncotarget

Article Title: Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

doi:

Figure Lengend Snippet: (A) Monoclonal antibodies were screened using flow cytometry to detect EphB4 expression on MCF-7 cells. The shift in peak fluorescence to the right (black filled) is compared with the matched isotype control (mIgG1, mIgG2a or rIgG) (red line) and is due to antibody binding. The H200 was used for comparison and the BerEP4 antibody that detects the epithelial glycoprotein EpCam was used as the positive control. (B) Western blot analysis showing that denatured and reduced EphB4 (120 kDa) can be identified using antibodies 13A7, 13B11 and 2D9. The Life Technologies “Zymed” monoclonal antibody was used as the positive control. (C) EphB4 was immunoprecipitated from total protein lysates using several of the monoclonal antibodies. EphB4 was identified in the immunoprecipitated sample via Western blot analysis using the Zymed antibody. (D) Immunofluorescence comparing EphB4 detection (green) in the MCF10A and MCF10A-B4 cells using the monoclonal antibodies. Bar = 80 μm.

Article Snippet: Three commercially available human EphB4-specific polyclonal antibodies EphB4 (N-19), EphB4 (H-200) and EphB4 (C-16) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Bioprocessing, Flow Cytometry, Expressing, Fluorescence, Control, Binding Assay, Comparison, Positive Control, Western Blot, Immunoprecipitation, Immunofluorescence

Journal: Oncotarget

Article Title: Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

doi:

Figure Lengend Snippet: (A) Each antibody was tested for their ability to prevent tubular network formation of MDA-MB-231 cells grown on Matrigel. C2 was the most effective in duplicate wells. (B) Tumour growth data for MDA-MB231 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Doxorubicin™ (green triangles) or untreated (PBS) control group (blue diamonds). The C2 monoclonal antibody significantly reduces the size of tumours by 18 days ( p < 0.001) and even performs better than Doxorubicin™ over this time course. (C) Tumour growth data for PC3 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Taxol (green triangles) or untreated (PBS) control group (blue diamonds). The C2 antibody does not affect PC3 tumour growth. (D) Immunofluorescence localization of EphB4 (green) in MDA-MB-231 cells using the C2 antibody. Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Bar = 10 μm. (E) Immunofluorescence localization of EphB4, using the C2 antibody (green), and the endoplasmic reticulum marker calnexin (red) in PC3 cells. DAPI (blue) stains the nucleus. Bar = 10 μm.

Article Snippet: Three commercially available human EphB4-specific polyclonal antibodies EphB4 (N-19), EphB4 (H-200) and EphB4 (C-16) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Positive Control, Control, Immunofluorescence, Marker

Journal: BMC Cancer

Article Title: EphB4 as a therapeutic target in mesothelioma

doi: 10.1186/1471-2407-13-269

Figure Lengend Snippet: EphB4 overoverexpression in MPM. ( A ) A panel of MPM tissues was stained with EphB4-specific antibody MAb131, and EphB4 expression level was scored and summarized. Moderate and strong expression was considered overexpression. ( B ) Representative images demonstrating EphB4 expression patterns in epithelioid and sarcomatoid subtypes. ( C ) 293T cells grown on 8-well chamber slide were transfected with human EphB4 overexpresion vector or empty vector pCDNA3.1. 2 days after transfection, cells were fixed with 4% paraformaldehyde and stained with MAb131 that was also used for staining in ( A ). Nuclei were counter-stained with DAPI.

Article Snippet: EphB4-specific antibody (MAb131) was produced by VasGene Therapeutics Inc. Bevacizumab (Genentech Inc) was purchased.

Techniques: Staining, Expressing, Over Expression, Transfection, Plasmid Preparation