ephb4  (Bio-Techne corporation)

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: Primer sequences used to amplify EphB4 and the control housekeeping genes PBGD and HPRT 1 with expected size of the corresponding PCR product in base pairs (bp) and the annealing temperature used in the reaction.

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques:

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: RT-PCR analysis of EphB4 expression in prostate cancer cell lines . RT-PCR analysis of EphB4 and PBGD expression in duplicate RNA samples from the three prostate cancer cell lines LNCaP, DU145 and PC3. Sizes of the amplified products are shown on the right (bp). An RNA sample to which no reverse transcriptase was added and a PCR reagent only (containing no template) amplification were also performed as negative controls for each stage of the RT-PCR.

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: Normalisation of EphB4 expression to housekeeping genes PBGD and HPRT1 . EphB4 expression normalised to PBGD in prostate cancer cell lines LNCaP, DU145 and PC3. (A) Triplicate amplifications of EphB4 and PBGD in three samples for each cell line were analysed to determine the relative levels of EphB4 expression using the BioRad iCycler. All EphB4 / PBGD ratios were close to 1 indicating that there are comparable amounts of both templates in each RNA samples. (B) Dilutions of a single RNA sample for each cell line were also amplified in triplicate with both gene primer sets to confirm that the ratio was consistent regardless of starting template concentration. (C) Triplicate amplifications of EphB4 , PBGD and HPRT1 in three samples for each cell line using the Corbett Rotorgene confirmed the previous result for EphB4 / PBGD and showed a similar result when EphB4 expression was normalised to a second housekeeping gene.

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques: Expressing, Amplification, Concentration Assay

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: Western analysis of EphB4 in prostate cancer cell lines . Western analysis of 20 μg EphB4 protein in prostate cancer cell lines LNCaP (L), DU145 (D) and PC3 (P). (A) The immunoblot was incubated sequentially with antibodies specific to EphB4, α-actin and calnexin and exposed to autoradiographic film for the indicated times. MCF10A engineered to express EphB4 (5 μg protein lysate) was used as a positive control. The arrow indicates the expected size of each protein – 120 kDa for EphB4, 43.2 kDa for α-actin and 90 kDa for calnexin. The size of the marker proteins is shown on the left in kDa. (B) Coomassie stained duplicate gel for loading comparison.

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques: Western Blot, Incubation, Positive Control, Marker, Staining

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: Immunofluorescent staining of EphB4 in prostate cancer cell lines . Immunofluorescent staining of EphB4 in prostate cancer cell lines showing diffuse staining on the surface and in the cytoplasm of LNCaP and DU145 and in the cytoplasm only of PC3. There was no reactivity to the primary antibody (EphB4 1° only) or secondary antibody (2° only) alone and little background fluorescence (No staining).

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques: Staining, Fluorescence

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: Immunohistochemical staining of EphB4 in prostate cancer samples . The expression of EphB4 in human prostate carcinomas was detected using immunohistochemical staining of formalin fixed, paraffin-embedded tissue samples. Images (20× magnification) of normal (panel 2) and diseased tissue (panel 4) from eight different patient samples are shown with a Haematoxylin/eosin stained consecutive section (panels 1 and 3). The Gleason score for each disease focus is shown in the top left-hand corner of the image of each tumour focus. A single sample that contained a focus of basal cell hyperplasia, two samples with well-differentiated adenocarcinoma, two samples of moderately differentiated adenocarcinoma and three samples of poorly differentiated adenocarcinoma are shown. The brown stain from the biotinylated secondary antibody indicates the EphB4 protein. Nuclei are stained with Harris haematoxylin and appear blue. Staining of normal tissues was either absent or weak and diffuse. Increased staining of the tumour tissue appeared to correlate with increased Gleason score. There was no cross-reactivity with the secondary antibody alone (result not shown).

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques: Immunohistochemical staining, Staining, Expressing, Formalin-fixed Paraffin-Embedded

Journal: BMC Cancer

Article Title: Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

doi: 10.1186/1471-2407-5-119

Figure Lengend Snippet: Immunohistochemical staining of EphB4 in several foci of a single prostate cancer sample . The expression of EphB4 a different foci in a single patient sample was compared by immunohistochemistry. Images from 10× magnification (A) and a region from this (as indicated by the box) at 20× magnification (B) are shown for 2 regions of normal prostate duct (1 and 2) and 8 regions with disease (3–10) from this single sample. A comparable level of EphB4 staining was seen in regions of high grade PIN (eg. focus 8 indicated PIN) and regions of poorly differentiated adenocarcinoma (eg. focus 7 indicated PDA). Regions of well-differentiated (eg focus 3) and moderately differentiated (eg focus 10) also show comparable staining.

Article Snippet: EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Biomedicines

Article Title: Eph/Ephrin Promotes the Adhesion of Liver Tissue-Resident Macrophages to a Mimicked Surface of Liver Sinusoidal Endothelial Cells

doi: 10.3390/biomedicines10123234

Figure Lengend Snippet: EphA2, ephrin-A1, EphB4, and ephrin-B1 localization in LSECs and Kupffer cells. Immunofluorescence micrographs showing EphA2, ephrin-A1, EphB4, and ephrin-B1 immunoreactivity (red) in CD146-positive LSECs (green) and F4/80-positive Kupffer cells (green) in the mouse liver. Cryostat sections were stained with the indicated antibodies and DAPI (blue). Green fluorescence images were merged with red, and/or blue fluorescence images in the same field. EphA2, ephrin-A1, EphB4, and ephrin-B1 immunoreactivity was localized in CD146-positive LSECs and weakly localized in F4/80-positive Kupffer cells (arrows). M, marginal region of the hepatic lobules; Pc, pericentral region of the hepatic lobules.

Article Snippet: Goat polyclonal antibodies against EphA2 (AF639), EphB4 (AF446), and ephrin-B1 (AF473) (R&D Systems, Minneapolis, MN, USA) and rabbit polyclonal antibody against ephrin-A1 (SAB4500696; Sigma-Aldrich) were used.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Biomedicines

Article Title: Eph/Ephrin Promotes the Adhesion of Liver Tissue-Resident Macrophages to a Mimicked Surface of Liver Sinusoidal Endothelial Cells

doi: 10.3390/biomedicines10123234

Figure Lengend Snippet: Expression of Ephs and ephrins in the mouse liver, liver Mø propagated using mixed culture, and LSECs. ( A ) The protein expression and tyrosine phosphorylation (PTyr) of EphA2 and EphB4 were detected using Western blotting in the mouse liver. EphA2 and EphB4 in the liver are highly and weakly tyrosine-phosphorylated, respectively. IP, immunoprecipitation. ( B , C ) RT-PCR analysis of all Ephs and ephrins in liver Mø propagated using mixed culture ( B ) and primary LSECs ( C ). Liver Mø express Epha2 , Epha4 , Efna1 , Efna4 , Efna5, Ephb3 , Ephb4 , Ephb6 , Efnb1 , Efnb2 , and Efnb3 whereas LSECs express Epha2 , Efna1 , Ephb4 , Efnb1 , Efnb2 , and Efnb3 .

Article Snippet: Goat polyclonal antibodies against EphA2 (AF639), EphB4 (AF446), and ephrin-B1 (AF473) (R&D Systems, Minneapolis, MN, USA) and rabbit polyclonal antibody against ephrin-A1 (SAB4500696; Sigma-Aldrich) were used.

Techniques: Expressing, Phospho-proteomics, Western Blot, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction