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: eLife

Article Title: High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display

doi: 10.7554/eLife.82345

Figure Lengend Snippet: ( A ) Heatmaps depicting the specificities of c-Abl, Fer, EPHB1, and EPHB2. Enrichment scores were log 2 -transformed and are displayed on a color scale from blue (disfavored sequence features, negative value), to white (neutral sequence features, near zero value), to red (favored sequence features, positive value). Values in the heatmaps are the average of three replicates. ( B ) Sequences of consensus peptides identified through X 5 -Y-X 5 screens, compared with previously reported SrcTide and AblTide sequences. ( C ) Phosphorylation kinetics of five consensus peptides against five kinases. Initial rates were normalized to the rate of the cognate consensus peptide. All peptides were used at a concentration of 100 μM, and the kinases were used at a concentration of 10–50 nM. Error bars represent the standard deviation from at least three measurements. Figure 2—source data 1. Position-specific amino acid enrichment matrices from the tyrosine kinase X 5 -Y-X 5 library screens. Matrices calculated with and without inclusion of multi-tyrosine sequences are provided.

Article Snippet: Recombinant DNA reagent , pET-His6-TEV-EPHB1(KD) , PMID: 30004690 , Addgene: 79694 , bacterial expression vector encoding the human EPHB1 kinase domain (residues 602–896) with an N-terminal His6-tag and TEV protease recognition sequence.

Techniques: Transformation Assay, Sequencing, Concentration Assay, Standard Deviation

Journal: eLife

Article Title: High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display

doi: 10.7554/eLife.82345

Figure Lengend Snippet: Cells displaying the X 5 -Y-X 5 library were treated with a kinase cocktail containing c-Src, c-Abl, AncSZ, and EPHB1 for 3 hr, then labeled with a pan-phosphotyrosine antibody (PY20 PerCP-eFluor 710) and analyzed by flow cytometry.

Article Snippet: Recombinant DNA reagent , pET-His6-TEV-EPHB1(KD) , PMID: 30004690 , Addgene: 79694 , bacterial expression vector encoding the human EPHB1 kinase domain (residues 602–896) with an N-terminal His6-tag and TEV protease recognition sequence.

Techniques: Labeling, Flow Cytometry

Journal: eLife

Article Title: High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display

doi: 10.7554/eLife.82345

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pET-His6-TEV-EPHB1(KD) , PMID: 30004690 , Addgene: 79694 , bacterial expression vector encoding the human EPHB1 kinase domain (residues 602–896) with an N-terminal His6-tag and TEV protease recognition sequence.

Techniques: Clone Assay, Over Expression, Strep-tag, Recombinant, Plasmid Preparation, Sequencing, Variant Assay, Expressing, Mutagenesis, Marker, Amplification, Synthesized, Western Blot, Purification, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Partial Inhibition of Glycolysis Reduces Atherogenesis Independent of Intraplaque Neovascularization in Mice

doi: 10.1161/ATVBAHA.119.313692

Figure Lengend Snippet: 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) stimulates autophagosome formation in endothelial cells independent of mTOR. A , Human aortic endothelial cells (HAOECs) were stimulated with 3PO (5–20 µmol/L) for 16 h followed by Western blot analysis for the autophagosomal marker protein LC3. Protein levels of LC3-I and LC3-II were quantified relative to the reference protein β-actin. * P <0.05 vs untreated control (ctrl) cells (1-way ANOVA, followed by Dunnett test, n=3). B , Detection and quantification of autophagic vacuoles (arrows) in 3PO-treated HAOECs (20 µmol/L 3PO, 16 h) using transmission electron microscopy. Scale bar=500 nm. *** P <0.001 (Mann-Whitney U test; n=3). C , Evaluation of the phosphorylation status of mTOR and its downstream target p70S6K in 3PO-treated HAOECs via Western blotting. Everolimus (EV; 10 µmol/L) was used as a positive control. LC3 indicates microtubule-associated protein light chain 3; and mTOR, mammalian target of rapamycin.

Article Snippet: The following primary antibodies were used: anti–β-actin (ab8226; Cell Signaling), anti-mTOR (2972; Cell Signaling), anti–phospho-mTOR (S2448; 2971; Cell Signaling), anti-p70 S6 kinase (9202; Cell Signaling), anti–phospho-p70 S6 kinase (Thr389; 9205; Cell Signaling), anti-LC3 (clone 5F10, 0231-100/LC3-5F10; Nanotools), anti–VCAM-1 (ab134047; Abcam), anti-ATG7 (8558S; Cell Signaling), anti–NF-κB (8242; Cell Signaling), anti-phospho–NF-κB (3039; Cell Signaling) and anti–ICAM-1 (ab179707; Abcam).

Techniques: Western Blot, Marker, Transmission Assay, Electron Microscopy, MANN-WHITNEY, Positive Control