Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 protein levels are increased in the retinas of diabetic patients and diabetic mice. Western blotting was done for EphB1 in whole retinal lysates from (A) control and diabetic patients and (B) control and diabetic mice. All diabetic patients had had the disease for over 10 years. *p<0.05 versus control. n=7 for both panels. Data are presented as mean ± standard error of mean (SEM).

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Western Blot, Control

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 is increased in rMC-1 Müller cells. Panel A shows the localization of EphB1 in rMC-1 cells in culture in normal glucose (top) and high glucose (bottom). Cells are stained with green for EphB1 and DAPI for nuclear localization. Panel B is western blot data from rMC-1 cells grown in normal (5 mM) or high (25 mM) glucose. *p<0.05 versus normal glucose (NG), n=5 for the western blot. Data are presented as mean ± SEM. Scale bar is 25 µm.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Staining, Western Blot

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 siRNA reduced inflammatory mediators in rMC-1 cells. rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose. Some cells in high glucose were treated with EphB1 siRNA or scrambled siRNA. Western blotting was performed for (A) EphB1, (B) TNFα, and (C) NLRP3. An ELISA was done for IL-1β in panel D. *p<0.05 versus NG, #p<0.05 versus high glucose (HG). n=5. Data are presented as mean ± SEM.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: Ephrin B1 increased inflammatory mediators in rMC-1 cells. rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose. Some cells in each glucose condition were treated with ephrin B1-Fc. Western blotting was performed for (A) HMGB1 and (B) NLRP3. An ELISA was done for IL-1β. *p<0.05 versus NG, #p<0.05 versus HG. n=5. Data are presented as mean ± SEM.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 can be expressed by AAV in Müller cells. To express EphB1 in retinal Müller cells in mice, mice were injected equal amounts of either the EphB1 overexpression AAV or control AAV. The left panel (green) shows HA staining (as a control), the middle panel is EphB1 (red), and the merged image is on the right with yellow staining demarcated by arrows showing co-localization. Five mice in each group were used for the staining experiments. Scale bar is 50 nm.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Injection, Over Expression, Control, Staining

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: Ischemia/reperfusion damage was worsened by overexpression of EphB1 by AAV. The top panels show retinal thickness and cell loss in the ganglion cell layer. Quantification of cell loss and retinal thickness is shown in graphs below the images. *p<0.05 versus AAV control, #p<0.05 versus AAV control I/R. n=5 for all groups. Data are mean ± SEM.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Over Expression, Control

Journal: The Journal of Neuroscience

Article Title: Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb

doi: 10.1523/JNEUROSCI.2863-20.2021

Figure Lengend Snippet: Antibodies and Fc reagents used

Article Snippet: EphB1 , Goat , 1:500 , Santa Cruz Biotechnology.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb

doi: 10.1523/JNEUROSCI.2863-20.2021

Figure Lengend Snippet: Paxillin function is specifically required for ephrin-mediated guidance of cultured LMC neurites. Growth preference on protein stripes exhibited by medial or lateral LMC axons. Each experiment is composed of three panels (left, middle, and right) and one quantification. A–H, Left panels, Detection of medial (GFP+) LMC neurites of explants on eB2/Fc (A), eB1/Fc (C), EB1/Fc (E), or N/Fc (G) stripes, and [paxillin]siRNA coelectroporated explants on eB2/Fc (B), eB1/Fc (D), EB1/Fc (F), or N/Fc (H) stripes. Middle panels, Inverted images where GFP signals are dark pixels overlaid on substrate stripes. Right panels, Superimposed images of five representative explants from each experimental group, highlighting the distribution of medial LMC neurites. Quantification of medial (GFP+) LMC neurites on first (pink) and second (pale) stripes expressed as a percentage of total GFP signals. Minimal numbers of neurites, 90. Numbers of embryos: n = 5 for all groups. Minimal numbers of explants per embryo, 3. I–P, Left panels, Detection of lateral (GFP+ EphA4+) LMC neurites of explants on eA5/Fc (I), eA2/Fc (K), EA2/Fc (M), or N/Fc (O) stripes, and [paxillin]siRNA coelectroporated explants on eA5/Fc (J), eA2/Fc (L), EA2/Fc (N), or N/Fc (P) stripes. Middle panels, Inverted images where EphA4 signals are dark pixel overlaid on substrate stripes. Right panels, Superimposed images of five representative explants from each experimental group, highlighting the distribution of lateral LMC neurites. Quantification of lateral (EphA4+) LMC neurites on first (pink) and second (pale) stripes expressed as a percentage of total EphA4 signals. Minimal numbers of neurites, 88. Numbers of embryos: n = 5 for all groups. Minimal numbers of explants per embryo, 3. eB2, Ephrin-B2-Fc; eB1, ephrin-B1-Fc; EB1, EphB1-Fc; eA5, ephrin-A5-Fc; eA2, ephrin-A2-Fc; EA2, EphA2-Fc; N, Netrin-1. ***p < 0.001; **p < 0.01; n.s. = not significant; statistical significance was computed using Mann–Whitney U test; solid lines in each scatterplot show the median difference. Scale bars: H (for A–H), P (for I–P), 150 μm.

Article Snippet: EphB1 , Goat , 1:500 , Santa Cruz Biotechnology.

Techniques: Cell Culture, MANN-WHITNEY

Journal: Journal of Cancer

Article Title: Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

doi: 10.7150/jca.44576

Figure Lengend Snippet: Clinical characteristics and EphB1 expression in 60 cases of NSCLC.

Article Snippet: Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

doi: 10.7150/jca.44576

Figure Lengend Snippet: Primer sequence for real-time PCR

Article Snippet: Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech.

Techniques: Sequencing

Journal: Journal of Cancer

Article Title: Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

doi: 10.7150/jca.44576

Figure Lengend Snippet: EphB1 expression is correlated with poor patient survival in lung cancer. The expression of EphB1 was analyzed in patients with NSCLC. (A) EphB1 expression in NSCLC samples and non-cancer controls; the publicly accessible gene expression data of EphB1 was obtained from Gene Expression Ominibus (GEO) database (GSE10072, GSE19188, GSE7670) and The Cancer Genome Atlas (TCGA). (B) EphB1 expression in NSCLC patients with or without metastasis and normal controls or tumor samples; The publicly accessible gene expression data of EphB1 was obtained from Gene Expression Ominibus (GEO) database (GSE68465, GSE30219, GSE50081) and The Cancer Genome Atlas (TCGA). Data were analyzed with Student's t-test, p values were shown. (C) Kaplan-Meier overall survival curves according to EphB1 expression in patient cohorts in GEO datasets. The percentage of survival patients in high EphB1 and low EphB1 groups at different time points are presented. p values were shown. (D) EphB1 expression in lung cancer patient with or without metastasis was measured by immunohistochemical staining. Scale bar: Above=100 µm; Below=20 µm; Data were analyzed with Student's t-test, *p=0.0143.

Article Snippet: Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

doi: 10.7150/jca.44576

Figure Lengend Snippet: Ligand-independent EphB1 signaling promotes cancer cell migration and invasion. (A) EphB1 expression in NSCLC cell lines. (B) (C) Transwell assay was conducted to test the effect of EphB1 forced expression and (D) Ligand EphrinB2-Fc treatment on the migration and invasion of NSCLC cells. Number of cells were counted and shown in the column graph on the right of the corresponding pictures. Data are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01. (E) The proliferation of NSCLC was measured by CCK-8 or (F) Clonogenic assay after expression of EphB1 or knockdown of EphB1. Number of cell clones were counted and shown in the column graph on the right side. Data are mean ± SD of three independent experiments. ns: no significance.

Article Snippet: Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech.

Techniques: Migration, Expressing, Transwell Assay, CCK-8 Assay, Clonogenic Assay, Knockdown, Clone Assay

Journal: Journal of Cancer

Article Title: Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

doi: 10.7150/jca.44576

Figure Lengend Snippet: Ligand-dependent EphB1 signaling inhibits cancer cell migration and invasion through inducing the phosphorylation of EphB1. (A) Western blot was conducted to test the phosphorylation of EphB1 after transfection of EphB1 or treatment of EphrinB2-Fc; (B) Transwell assay was conducted to test the effect of EphB1 wt and mutant upon treatment of ligand EphrinB2-Fc on the migration and invasion of NSCLC cells. Number of cells were counted and shown in the column graph on the right of the corresponding pictures. Data are mean ± SD of three independent experiments. ** P < 0.01, *** P < 0.001, ns: no significance.

Article Snippet: Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech.

Techniques: Migration, Phospho-proteomics, Western Blot, Transfection, Transwell Assay, Mutagenesis

Journal: Journal of Cancer

Article Title: Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration

doi: 10.7150/jca.44576

Figure Lengend Snippet: Ligand-independent EphB1 mediates TGF-β-activated N-cadherin. (A) Expression of mesenchymal molecules in lung cancer cells after transfection of EphB1 or knockdown of EphB1 quantified by real-time PCR. *p<0.05; **p<0.01; ***p<0.001. (B) Expression of CDH2 upon transfection of EphB1 measured by Western blot. (C) Expression of EphB1 upon treatment of TGF-β measured by Western blot. (D) Western blot was conducted to test the expression EphB1 after transfection of Smad2 or knockdown of Smad2. (E) Expression of EphB1 after transfection of Smad2 or knockdown of Smad2 quantified by real-time PCR. (F) The putative Smad2/3 binding sites in EphB1 promoter. (G) ChIP assay in 293 cells to detect the binding of Smad2 to the promoter of EphB1. Mean values± SD of three independent experiments are shown on the right side. IgG indicates nonspecific Ab.

Article Snippet: Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; N-cadherin (#13116) and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig), Smad2 (#12570-1-AP) and GAPDH (#60004-1-Ig) were from Proteintech.

Techniques: Expressing, Transfection, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Targeting the Eph/Ephrin System as Anti-Inflammatory Strategy in IBD

doi: 10.3389/fphar.2019.00691

Figure Lengend Snippet: Purported functional effects of administered recombinant proteins ephrin-B1-Fc, EphB1-Fc, and EphB4.

Article Snippet: Briefly, 96-well ELISA high binding plates (Costar, Corning, New York, NY, USA 9018) were incubated overnight at 4°C with 100 μL/well of 1 μg/mL EphB1-Fc (R&D systems, Minneapolis, MN, USA, 1596-B1) or EphB4-Fc (R&D systems, Minneapolis, MN, USA, 446-B4) diluted in sterile phosphate buffered saline (PBS—0.2 g/L KCl, 8.0 g/L NaCl, 0.2 g/L KH 2 PO 4 , 1.15 g/L Na 2 HPO 4 , pH 7.4).

Techniques: Functional Assay, Recombinant

Journal: Frontiers in Pharmacology

Article Title: Targeting the Eph/Ephrin System as Anti-Inflammatory Strategy in IBD

doi: 10.3389/fphar.2019.00691

Figure Lengend Snippet: Experimental design with characteristics of each experimental group.

Article Snippet: Briefly, 96-well ELISA high binding plates (Costar, Corning, New York, NY, USA 9018) were incubated overnight at 4°C with 100 μL/well of 1 μg/mL EphB1-Fc (R&D systems, Minneapolis, MN, USA, 1596-B1) or EphB4-Fc (R&D systems, Minneapolis, MN, USA, 446-B4) diluted in sterile phosphate buffered saline (PBS—0.2 g/L KCl, 8.0 g/L NaCl, 0.2 g/L KH 2 PO 4 , 1.15 g/L Na 2 HPO 4 , pH 7.4).

Techniques: Saline