rabbit monoclonal anti py588 epha2  (R&D Systems)

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: EPHA2 is expressed on the cell surface of undifferentiated mouse ESCs and downregulated during differentiation. (A) Relative gene expression of Epha2 mRNA in mouse ESCs (D3) cultured without LIF for 0, 3, 5, and 7 days. (B) Immunoblotting of EPHA2 protein of mouse ESCs cultured as in (A). (C) EPHA2 protein levels in (B) normalized to α-Tubulin. (D) Immunofluorescent staining of mouse ESCs. The cells were cultured with or without LIF for 7 days. Bars; 200 μm. (E, F) Flow cytometric analysis of EPHA2 protein on the cell surface of mouse ESCs. Living cells were stained with an EPHA2 antibody. The propidium iodide + dead cells were removed from the analysis. FSC and SSC profiles (E) and the histogram of EPHA2-AF488 levels (F) of the gated cells in (E) were shown. Statistical analyses in (A) and (C) were calculated by Dunnett’s test. Data were graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01; *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Gene Expression, Cell Culture, Western Blot, Staining

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Knock-down of Epha2 induces spontaneous differentiation of mouse ESCs. (A) qRT-PCR analysis of Epha2 mRNA after knock-down in mouse ESCs (D3). The cells were infected with Epha2 shRNA retrovirus and cultured with or without LIF for 7 days. (B) Images of phase contrast and alkaline phosphatase (AP) staining of Epha2 KD mouse ESCs cultured in ES maintenance medium with LIF for 5 days. Bars; 200 μm. (C) qRT-PCR analysis of undifferentiated state-specific marker genes in Epha2 KD mouse ESCs. (D) Immunofluorescent staining of Epha2 KD mouse ESCs cultured with LIF for 7 days. Bars; 100 μm. (E) Culture conditions of Epha2 KD mouse ESCs after infection with Epha2 shRNA retrovirus. One day after infection with Epha2 shRNA virus, mouse ESCs were cultured with or without 2i in the presence of G418 and LIF for 4 days. (F) Phase contrast images of Epha2 KD mouse ESCs cultured as in (E). Bar; 200 μm. (G) qRT-PCR analysis of Epha2 and undifferentiated state-specific marker genes in Epha2 KD mouse ESCs cultured with 2i. All qRT-PCR analyses were performed with 3 independent biological replicates and graphed as means ± SE. The significant differences were calculated by Tukey test in (A and C) and t -test in (G). * P < .05; ** P < .01, *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Knockdown, Quantitative RT-PCR, Infection, shRNA, Cell Culture, Staining, Marker, Virus

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Expression of EPHA2 in heterogenous subpopulation of human PSCs. (A) qRT-PCR analysis of EPHA2 and undifferentiated state-specific marker genes during EB-based random differentiation of human iPSCs (201B7) without basic FGF. Statistical analysis was done by Dunnett’s test comparing to day 0 and graphed as means ± SE of 3 independent experiments. * P < .05; ** P < .01. (B) Representative flow cytometric plots of living human iPSCs stained with EPHA2 antibody-conjugated with AFF488. Gate3 and Gate4 were sorted as EPHA2 − and EPHA2 + cell populations, respectively. See also . (C) Feature plots of EPHA2 and undifferentiated state-specific genes in publicly available undifferentiated human ESC H1 and H9 data subsets from GSE75748 scRNA-seq dataset. Note that EPHA2 expression in hESCs was heterogeneous. Normalized expression levels were plotted. (D, E) Immunofluorescent staining of human iPSC cultured on SyntheMax II-coated plate with StemFit medium. The cells were fixed with paraformaldehyde in PBS and permeabilized. Bars; 200 μm. (F) qRT-PCR analysis of fractioned EPHA2 + and EPHA2 - subpopulations. EPHA2 + cells express higher OCT4 and NANOG , than EPHA2 − cells. Means ± SE of 3 independent experiments were shown. Statistical significance was defined as * P < .05 by t -test.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Quantitative RT-PCR, Marker, Staining, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Transplantation of EPHA2 + cells into immune-deficient mice formed tumors in vivo. (A) Immunofluorescent staining of mouse EBs differentiated into hepatocyte lineages. Expression of an early hepatocyte marker AFP at day 10 and a mature marker ALB at day 14 were detected. Bars; 200 μm. (B) Depletion of undifferentiated ES colonies after removal of EPHA2 + cells from EBs. Oct4-egfp ESCs were differentiated by EB formation for 10 and 14 days. EPHA2 + cells were removed from EBs using anti-EPHA2 antibody-bound MACS after dissociation with trypsin/EDTA. The residual cells were cultured in ES maintenance medium with LIF for 7 days. The alkaline phosphatase (AP) activity was visualized by incubating with AP substrate. Bar; 2 cm. (C) The number of EGFP + cell colonies in (B). Statistical analysis was done by Tukey test and graphed as means ± SE of 4 independent experiments. (D) Scheme of in vivo transplantation experiment. Mouse ESCs (D3) were differentiated into hepatocyte linages and the EBs were dissociated by EDTA treatment. The cells were transplanted into SCID mice after depletion of EPHA2 + cells by MACS. (E) Decreased teratoma formation after transplantation of EPHA2 − cells. White arrowheads indicate teratomas. (F) H&E staining of teratomas formed in the testicular subcutaneous tissue without MACS procedure. Typical cell types of 3 germ layers were confirmed. Bar; 500 μm. (G) Typical teratoma formation after transplantation of dissociated EB at day 10 through hepatic portal vein. White arrowheads indicate teratomas. (H) Quantification of teratoma formation in (E) and (G). Statistical analysis was done by Chi-square test, * P < .05, ** P < .01.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Transplantation Assay, In Vivo, Staining, Expressing, Marker, Cell Culture, Activity Assay

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Co-expression of EPHA2 with OCT4 in EBs during human iPSC differentiation into hepatocyte. (A, B) Relative gene expression of undifferentiation and differentiation markers during hepatic induction. Statistical analysis was done by Dunnett’s test against day 0 and graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01, *** P < .001. (C) Immunofluorescent staining of EBs at days 5, 8, and 10. Bars; 200 μm. (D) Enlarged images of EB at day 5 in (C). Bars; 50 μm. (E, F) Quantification of immune-positive cells in . Box plot showing the percentage of EPHA2 + cells among SOX17 + or OCT4 + cells (E). Box plot showing the percentage of EPHA2 + and TRA1-81 + cells among OCT4 + cells (F). Each box represents 1st quartile, median, and 3rd quartile, and whiskers show the minimum and maximum values. Ten images of independent EBs were analyzed. Total count of DAPI + nuclei per image were between 1 × 10 3 and 2 × 10 3 . Statistical significance was defined by Tukey test of SOX17 and OCT4, respectively in (E) and t -test between EPHA2 and TRA1-81 in (F). **P p < .01, *** P < .001, N.S; no significance between 3 with P > .05.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Gene Expression, Staining

Journal: Oncology letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade.

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of glioma tissues. (A) MMP‑2 and (B) EphA2 expression was identified by immunofluorescence staining in the cytoplasm of glioma cells. Positive cells were stained brown. Percentages of stained cells are presented in the bar graphs. Data represent the mean ± standard deviation (n=5). ***P<0.001. Scale bar, 30 µm. EphA2, ephrin type‑A receptor 2; MMP‑2, metalloproteinase 2.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25 ̊C for 1‐5 min. After blocking in BSA solution 37 ̊C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3'-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Immunohistochemical staining, Staining, Expressing, Immunofluorescence, Standard Deviation

Journal: Oncology letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade.

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Figure 3. Pearson's correlation analysis of the magnetic resonance imaging parameters and immunohistochemistry results. Scatterplots presenting the correla- tion between (A) MMP‑2 and (B) EphA2 positivity with the EI; (C) MMP‑2 and (D) EphA2 positivity with the EP; and (E) MMP‑2 and (F) EphA2 positivity with maximum tumor diameter. EI, edema index; EP, enhancement percentage; EphA2, ephrin type‑A receptor 2; MMP‑2, metalloproteinase 2.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25 ̊C for 1‐5 min. After blocking in BSA solution 37 ̊C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3'-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Magnetic Resonance Imaging, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Safety, tolerability, pharmacokinetics, and pharmacodynamics of the afucosylated, humanized anti-EPHA2 antibody DS-8895a: a first-in-human phase I dose escalation and dose expansion study in patients with advanced solid tumors

doi: 10.1186/s40425-019-0679-9

Figure Lengend Snippet: Patient characteristics

Article Snippet: EPHA2 was detected using anti-human EPHA2 mouse monoclonal antibodies (clones 018 and 058, Daiichi Sankyo Co., Ltd.).

Techniques: Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Safety, tolerability, pharmacokinetics, and pharmacodynamics of the afucosylated, humanized anti-EPHA2 antibody DS-8895a: a first-in-human phase I dose escalation and dose expansion study in patients with advanced solid tumors

doi: 10.1186/s40425-019-0679-9

Figure Lengend Snippet: Efficacy results

Article Snippet: EPHA2 was detected using anti-human EPHA2 mouse monoclonal antibodies (clones 018 and 058, Daiichi Sankyo Co., Ltd.).

Techniques: