Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand-binding domain and TK domain by us recently

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Amplification

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Control, Immunohistochemistry, Expressing, Staining

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Mutagenesis, Inhibition, Plasmid Preparation

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques:

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å 3 ) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Binding Assay

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: In vitro characterization of IgG25 and IgG28. (a) FACS-based whole cell binding assay was performed with IgG25 and IgG28 on human MiaPaCa2 and (b) on mouse MC38-CEA cells to determine the apparent Kd. Mean Fluorescence Intensities (MFIs) obtained with IgG25 and IgG28 over the logarithm of their molar concentration (LogM) are reported. (c) Binding competition experiments of IgG25 and IgG28 with ephrinA1 on MiaPaCa2 cells, a control isotypic IgG1 antibody (Ctrl IgG) was used as negative control. IgG25 (filled circles), IgG28 (empty circles), Ctrl IgG (filled triangles). (d) EphA2 immunoprecipitation from lysates of cells treated with Ctrl IgG, IgG25, IgG28 and ephA1-Fc, followed by Western Blot with antiphosphotyrosine antibody. After stripping, the same filter was probed with EphA2 antibody as loading control. (e) FACS-based whole cell binding with IgG25 and IgG28 on mouse N2A cells transiently transfected with expression vectors coding for members of Eph A receptor family (EphA1, EphA2, EphA3, EphA4, EphA5, and EphA7).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vitro, Cell Binding Assay, Fluorescence, Concentration Assay, Binding Assay, Control, Negative Control, Immunoprecipitation, Western Blot, Stripping Membranes, Transfection, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25- and IgG28-mediated EphA2 internalization and degradation in MiaPaCa2 cells. (a) EphA2 internalization in response to IgG25, IgG28, and ephrinA1-Fc treatment. mAbs and ephrinA1-Fc labeled in red while cell nuclei in blue. Images were acquired at 20× magnification. Localization was revealed 1 hour after incubation on cells either at 4°C or at 37°C. (b) Time course Western blot analysis of EphA2 degradation after treatment with control IgG, IgG25, IgG28, and ephrinA1-Fc; anti- β -actin was used as loading control. (c) Densitometric analysis of the level of EphA2 expression measured by Western Blot in cells treated with control IgG (asterisks), IgG25 (squares), IgG28 (triangles), and ephrinA1-Fc (circles). Data are expressed as percentage of EphA2 expression over time.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Labeling, Incubation, Western Blot, Control, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 downstream signaling in MiaPaCa2 cells treated with IgG25 and IgG28. Cell lysates of MiaPaCa2 were assayed by Western Blotting with antiphospho-Akt, antiphospho-ERK, and anti-phospho FAK (Tyr 576). Antibodies directed to total Akt, total FAK, and actin were used as loading controls.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Western Blot

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: Effect of EphA2 antibodies on angiogenesis. (a) Representative CD31 immunostaining of paraffin embedded tumor section (magnification bar = 20 m). (b) Quantitative analysis of CD31 staining in tumors treated with control IgG, IgG25, and IgG28. The average data obtained from the analyses of two tumors selected from each group are reported as the percentage of CD31 + area in each entire section. In all panels, asterisks indicate statistically significant differences with respect to the control group (Student's t -test; P <.05).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Immunostaining, Staining, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 protein expression in tumors from treated mice. (a) Western Blot analysis with anti-EphA2 antibodies of lysates from five tumors treated with control IgG, with IgG25, or six tumors treated with IgG28. (b) Densitometric analysis of the ratio between EphA2 levels revealed by anti-EphA2 antibody in nonsaturating conditions and actin expression measured with antiactin antibody. Data on y axis are expressed as arbitrary units.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25 and IgG28 modulate in vivo EphA2 downstream signaling: Western Blot analyses of tumor lysates with antiphospho FAK (Tyr 576) and anti-phospho Akt. Antibodies to total Akt, total FAK, and actin were used as loading controls. P values were calculated with respect to average densitometric value of group treated with control IgG.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vivo, Western Blot, Control