Journal: Chemical biology & drug design

Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

doi: 10.1111/j.1747-0285.2011.01199.x

Figure Lengend Snippet: Compound 76D10 inhibits EphA4 and EphA2 activation and cell retraction after ephrin stimulation. (A) Cells pretreated with the indicated concentrations of 76D10 for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compound. COS cells were stimulated with 0.2 μg/mL ephrin-A1 or 0.8 μg/mL ephrin-B2 Fc and used to immunoprecipitate EphA2 and EphB2, while HT22 neuronal cells were stimulated with 0.2 μg/mL ephrin-A1 and used to immunoprecipitate EphA4. Eph immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for the Eph receptor immunoprecipitated. (B) PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.2 μg/mL ephrin-A1 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. The histogram shows the average level of phosphorylated EphA2 normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. Error bars represent standard errors from 4–10 measurements. The levels of EphA2 phosphorylation in cells treated with ephrin-A1 Fc and compound were compared to those in cells treated only with ephrin-A1 Fc by one-way ANOVA and Dunnett’s post test. ***P<0.001 by one-way ANOVA. (C–D)PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.5 μg/ml ephrin-A 5Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. (C) The histogram shows the average area of the cells normalized to the value obtained for the Fc-treated cells. Error bars represent standard errors from three wells. The average cell areas in cells treated with ephrin-A1 Fc and compound were compared to that in cells treated only with ephrin-A1 Fc by one-way ANOVA and Bonferroni’s post test, showing that 76D10 significantly (***P<0.001) inhibits ephrin-A1-dependent cell retraction at concentrations between 100 and 25 μM. The effect of ephrin-A1 was reverted completely by 100 μM 76D10 and partially by 50 and 25 μM (comparison between Fc and ephrin-A1 Fc treated samples at each compound concentration yielded P values of >0.05 for 100μM, <0.05 for 50 μM and <0.001 for 25μM 76D10. (D) Representative images of cells stained with rhodamine-phalloidin to label actin filaments (red) and DAPI to label nuclei (blue). Scale bar = 50 μm.

Article Snippet: Polystyrene high binding capacity plates (Corning, Corning, NY) were incubated overnight at 4°C with 4 μg/ml goat anti-EphA2 antibody (directed to the extracellular region of the receptor; R&D Systems, Minneapolis, MN) diluted in phosphate buffered saline (PBS), and then incubated for 2 hours at room temperature with cell lysate diluted in RIPA or ELISA lysis buffer.

Techniques: Activation Assay, Control, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Comparison, Concentration Assay, Staining

Journal: Chemical biology & drug design

Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

doi: 10.1111/j.1747-0285.2011.01199.x

Figure Lengend Snippet: Compound 76D10 inhibits ephrin- and TNFα-induced tyrosine phosphorylation and capillary-like tube formation in HUVE cells. (A) Cells plated on Matrigel were treated with the indicated concentrations of 76D10 or DMSO and imaged 18 hours later. The number of polygons present in each picture and the average tube length were quantified. The histograms show averages from 4 independent experiments and the error bars represent the standard errors. **P<0.01 and ***P<0.001 by one-way ANOVA and Dunnett’s post test. (B) HUVE cells were left unstimulated or stimulated with 20 nM TNFα for 2 hours in the presence of the indicated concentrations of 76D10. EphA2 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphA2. (C) MTT assay to determine the number of viable HUVE cells after growth in the presence of the indicated concentrations of 76D10 for 1 or 3 days. Only DMSO was used in the “0 μM” sample, as a control. The histogram shows average absorbance at 570 nm in the presence of 76D10 normalized to the absorbance in the absence of the compound. Error bars represent standard error from 3 measurements in each of two experiments. *P<0.05 by one-way ANOVA and Dunnett’s post test for the comparison to cells not treated with compound (0 μM).

Article Snippet: Polystyrene high binding capacity plates (Corning, Corning, NY) were incubated overnight at 4°C with 4 μg/ml goat anti-EphA2 antibody (directed to the extracellular region of the receptor; R&D Systems, Minneapolis, MN) diluted in phosphate buffered saline (PBS), and then incubated for 2 hours at room temperature with cell lysate diluted in RIPA or ELISA lysis buffer.

Techniques: Phospho-proteomics, MTT Assay, Control, Comparison

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A–C) Epha2 homozygous deletion in mice causes development of progressive cataract. (A) Cataracts were visible by gross inspection in homozygous Epha2 knockout mice ( Epha2 −/− ) between 5 to 8 months of age, but not in heterozygous or wild type mice. Shown are slit lamp images confirming development of cataract in Epha2 −/− but not Epha2 +/+ mice. (B) Dark field imaging of the dissected lens. Although not readily detectable by visual inspection, cataracts were found on dissected lens by 3 months of age. This lens was tilted to show denser opacity near the equator (arrow). Enucleation frequently occurred during dissection of mature cataract after 8 months (far right). (C) Retroillumination examination revealed clusters of small vacuoles by one month of age. Scale bars: 1 mm for middle panel; 150 µm for right panel. (D) Immunoblot of total lens lysates showing decreasing EPHA2 expression with aging. (E–M) Compartmentalized and gradient expression of EPHA2 (red) in mouse lens. Blue: DAPI nuclear staining. (E–I) Midsagittal sections of lens from 14-day-old wild type mice were stained for EPHA2. (E) Low power view of an entire lens revealed dense expression of EPHA2 in subcortical lens fiber cells. Dotted arrows indicate gradient expression in lens epithelial cells near the equator. Scale bar: 1 mm. (F) Low EPHA2 expression in anterior lens epithelial cells (arrow head, sandwiched between dotted lines). (G) Inset from (F) showing high EPHA2 expression in lens fiber cells. (H) High level of EPHA2 expression at the bow. (I) Inset from (H) showing dense expression at modulus (arrow). Scale bars: 5 µm for F–I. (J–M) Coronal sections through the bow region of lens co-stained for EPHA2 and N-cadherin. (J) Note the spatially regulated expression pattern in subcortical lens fiber cells. (K) Inset from (J) showing “honey-comb” membrane staining pattern of EPHA2 in the cross sections of fiber cells at high magnifications. (L) N-cadherin from the same section show overlapping but distinct expression pattern compared with that of EPHA2. (M) Merged images of EPHA2/N-cadherin. 10 µm for J, L, and M; 2 µm for K.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Knock-Out, Imaging, Dissection, Western Blot, Expressing, Staining, Membrane

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: Incidence of Visible Cataracts in Wild-Type and Epha2 -null Mice.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A–D) Expression of ephrin-A1, a ligand for EPHA2. Note the disorganized ephrin-A1 expression and formation of vacuoles in the Epha2 −/− lens (arrow heads). Scale bars: 1 mm for A and C; 10 µm for B and D. (E,F) N-cadherin staining showing disorganization of lens fiber cells. Scale bars: 5 µm. (G) Overexpression of HSP25 but not HSP90 in Epha2 −/− lens which was quantified in (H), and confirmed by immunofluorescence staining (I). Scale bars: 40 µm. (J) Immunoblot for phosphorylated HSP25 revealed relatively low degree of phosphorylation in Epha2 −/− lens.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Expressing, Staining, Over Expression, Immunofluorescence, Western Blot, Phospho-proteomics

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: Characteristics of SNPs in the EPHA2 gene.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: P values from rank transformed traits and effect sizes from the quantitative cortical scores (β) of the risk allele at markers under the dominant model in EPHA2 for each separate study and for the joint analysis of all studies.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Transformation Assay

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A) Examination of crystal structure of EPHA2 kinase domain reveals that Arg721 in αE forms a salt bridge with Asp872 in αI. (B) Concordant conservation of Arg721 and Asp872 in different members of human Eph kinases. Note that EPHA9 and EPHB5 are not present in human genome and are not shown. (C) The same residues are also concordantly conserved across different species.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques:

Journal: PLoS Genetics

Article Title: EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

doi: 10.1371/journal.pgen.1000584

Figure Lengend Snippet: (A,B) Arg721Gln mutation causes an increased basal activation of EPHA2 kinase, which was correlated with dramatically reduced basal ERK1/2 activities. In a kinetic study (A), HEK 293 cells expressing WT-, Arg721Gln -EPHA2 or vector control were stimulated with 2 µg/ml ephrin-A1-Fc for the indicated times. In a separate experiment, a dose-response study was carried out (B), where different doses of ephrin-A1-Fc were used to stimulate cells expressing WT- or Arg721Gln -EPHA2 for 10 min. Cell lysates from both experiments were blotted with the indicated antibodies as described previously . (C) HEK 293 cells expressing Arg721-Gln mutant EPHA2 but not WT-EPHA2 were growth-inhibited by ephrin-A1 in a clonal growth assay as described previously . About 200 cells/well were seeded in a 24-well culture dish and cultured for 10 days in the presence or absence of ephrin-A1. (D) Stochastic intracellular trapping of Arg721Gln mutant in MEF cells derived from Epha2 knockout embryos. Shown is a cluster of cells with the mutant EPHA2 trapped inside the cells. In contract, WT-EPHA2 was primarily expressed on the cytoplasmic membrane. Scale bar: 5 µm.

Article Snippet: Antibodies used include: goat anti-mouse EPHA2 ectodomain, goat anti-human EPHA2 (R&D Systems, Minneapolis, MN), rabbit anti-EPHA2 and anti-ephrin-A1, goat anti-HSP25 and mouse anti-phospho-ERK, rabbit anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-HSP25, anti-phospho-AKT, anti-Akt, anti-GAPDH (Cell Signaling), mouse monoclonal anti-N-cadherin (BD Biosciences).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Control, Growth Assay, Cell Culture, Derivative Assay, Knock-Out, Membrane

Journal: PLOS Pathogens

Article Title: Candida albicans translocation through the intestinal epithelial barrier is promoted by fungal zinc acquisition and limited by NFκB-mediated barrier protection

doi: 10.1371/journal.ppat.1012031

Figure Lengend Snippet: Detection antibodies for immune signaling proteins.

Article Snippet: p-EphA2 (S897) , Rabbit , 1:1,000 , Cell Signaling , 6347.

Techniques: