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 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:

Journal: Molecular cancer therapeutics

Article Title: Manipulation of cell-type selective antibody internalization by a guide-effector bispecific design

doi: 10.1158/1535-7163.MCT-18-1313

Figure Lengend Snippet: A) Illustration of the tetravalent ALCAMxEphA2 bsIgG. The IgG backbone is based on the non-internalizing anti-ALCAM antibody 3F1. The internalizing anti-EphA2 scFv is fused to the end of light chain C-terminus. B) Confocal microscopy study of antibody internalization. HEK293 or HEK293-EphA2#2 cells were incubated with indicated IgG or bsIgG (100 nM) at 37 °C for 2 hours. Antibodies (red) were detected using Alexa® 647-labeled anti-human IgG secondary antibody, and cell images were analyzed using a digital laser confocal microscope. Scale bar: 20 μm. C) Kinetics of ALCAM cell surface removal by the bispecific. HEK293-EphA2#2 cells were incubated with indicated IgG or bsIgG for 1, 4, and 24 hours and surface ALCAM levels determined by FACS. The non-internalizing ALCAM is removed from cell surface by the bispecific (3F1/RYR) but not monoclonal antibodies. D) Correlation between surface antigen (ALCAM) removal efficiency and EphA2/ALCAM (E/A) expression ratio. HEK293 cell models with varying EphA2/ALCAM ratios were incubated with 3F1, 3F1/RYR, and C10/RYR (all at 100 nM), and antigens remaining on the cell surface were determined by anti-ALCAM antibodies that bind to a different epitope than 3F1. Pearson’s correlation coefficient (r) was calculated (0.3266, −0.7550, and −0.1896 for 3F1, 3F1/RYR, and C10/RYR, respectively) and trend-lines were depicted according to linear regression analysis. Data represent mean ± SD (duplicate). E) Illustration depicting bispecific-induced ALCAM internalization when the guide to effector ratio > threshold. CM: cell membrane. F) Significant retardation of EphA2 internalization by the bispecific 3F1/RYR when guide to effector ratio falls below a threshold level. HEK293 cells that possess a low EphA2/ALCAM ratio (< 0.2) were incubated with indicated antibodies (100 nM), and surface EphA2 levels were measured by FACS. P values were determined using two-tailed Student’s t-test. *P<0.05, and ***P<0.001. G) Illustration of the phenomenon shown in panel F where EphA2 internalization is retarded when the EphA2 to ALCAM (E/A) ratio falls below a threshold.

Article Snippet: Stable EphA2-expressing clones were identified by FACS using human anti-EphA2 antibody followed by Alexa Fluor® 647-labeled goat anti-human IgG (Jackson ImmunoResearch).

Techniques: Confocal Microscopy, Incubation, Labeling, Microscopy, Expressing, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Manipulation of cell-type selective antibody internalization by a guide-effector bispecific design

doi: 10.1158/1535-7163.MCT-18-1313

Figure Lengend Snippet: A) ALCAM cell surface level post antibody treatment. Pancreatic cancer cell lines (L3.6pl, Capan-1, Panc-1) were incubated with 3F1, 3F1/RYR, C10/RYR, or a mixture of 3F1 and C10/RYR. Following wash post treatment, cell surface ALCAM level was determined using an Alexa® 647-labeled IgG that bind to a different epitope on ALCAM than 3F1. MFI values were normalized against cells without antibody treatment. **P<0.01, and ***P<0.001. Duplicates. B) Confocal microscopy study of cell-type selective internalization mediated by the bispecific. L3.6pl (E/A ratio > 0.2) and Panc-1 (E/A ratio < 0.2) cells were incubated with 3F1, 3F1/RYR, or C10/RYR, and internalizing antibodies were stained with FITC-labeled anti-human IgG. Scale bar: 20 μm. C) Co-localization of antibodies and macropinocytotic vesicles. L3.6pl cells were incubated with 3F1, 3F1/RYR, or C10/RYR at 100 nM and ND70-TR (TR-Dextran, red) for 2 hours. Antibodies were detected by FITC-labeled anti-human IgG (green). Nuclei were labeled with Hoechst 33342 (blue). Scale bar: 10 μm. D) Lysosomal trafficking post internalization. L3.6pl cells were incubated with indicated antibodies (100 nM) for 2 hours. Internalized antibodies (green) and nuclei (blue) were stained as described in C), and lysosomes were detected using rabbit anti-LAMP1 primary IgG, followed by Alexa® 647-labeled anti-rabbit IgG (red). Scale bar: 10 μm. E) Retarded EphA2 internalization on Panc-1 cell when targeted by the bispecific. **P<0.01, and ***P<0.001. Duplicates. F) A time course of EphA2 removal from Panc-1 cell surface at 0.5, 1, and 4 hours post antibody treatment.

Article Snippet: Stable EphA2-expressing clones were identified by FACS using human anti-EphA2 antibody followed by Alexa Fluor® 647-labeled goat anti-human IgG (Jackson ImmunoResearch).

Techniques: Incubation, Labeling, Confocal Microscopy, Staining

Journal: Molecular cancer therapeutics

Article Title: Manipulation of cell-type selective antibody internalization by a guide-effector bispecific design

doi: 10.1158/1535-7163.MCT-18-1313

Figure Lengend Snippet: A) Significant ALCAM upregulation on L3.6pl sphere cells compared to non-sphere tumor cells. Adherent- or sphere-cultured L3.6pl cells were separated into single cells and antigen expression was measured using 3F1 or RYR IgG, followed by Alexa® 647-labeled anti-human IgG. The EphA2/ALCAM ratio is ~ 0.15 for sphere and ~ 0.305 for monolayer cells. B) ALCAM removal from the surface of sphere-forming cells by 3F1/RYR. Single cell population of L3.6pl (200 cells/well) was incubated with indicated antibodies (100 nM) for 2 weeks in an ultra-low attachment well plate. Cell surface levels of ALCAM post antibody treatment were determined by FACS. MFI values were normalized against control (no antibody treatment). **P<0.01. Duplicates. C) Antibody internalization into L3.6pl spheres. Tumor spheres incubated with indicated antibodies were collected by centrifugation, fixed, and permeabilized for confocal microscopy analysis. Antibodies and nuclei were stained with Alexa® 647-labeled anti-human IgG (red) and Hoechst 33342 (cyan), respectively. Scale bar: 10 μm. Intracellular antibody fluorescence intensity was quantified by Image J and shown in the right panel. ***P<0.001. D) Inhibition of L3.6pl tumor sphere formation by 3F1/RYR – reduction in number. Tumor sphere numbers (> 100 μm) were counted 14 days post antibody treatment (left) with representative well images shown (right). Error bars represent SD of a duplicate. *P < 0.05. E) Inhibition of L3.6pl tumor sphere formation by 3F1/RYR – reduction in size. **P<0.01. Duplicates. Scale bar: 100 μm.

Article Snippet: Stable EphA2-expressing clones were identified by FACS using human anti-EphA2 antibody followed by Alexa Fluor® 647-labeled goat anti-human IgG (Jackson ImmunoResearch).

Techniques: Cell Culture, Expressing, Labeling, Incubation, Centrifugation, Confocal Microscopy, Staining, Fluorescence, Inhibition

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Confocal microscopic images of OKF6/TERT-2 epithelial cells that had been infected with GFP expressing C. albicans (CAI4-GFP) and then stained for EphA2 (red). Results are representative of 3 independent experiments. Arrows indicate the accumulation of EphA2 around the fungal cells. Hollow arrows indicate organisms that were analyzed for fluorescent intensity in ( b ). Negative control images are shown in ( b ) Magnified image of C. albicans cells with plots of fluorescent intensity at the regions indicated by the dotted line. The green lines indicate the fluorescent intensity of GFP expressing C. albicans and the red lines indicate the fluorescent intensity of the EphA2. ( c ) Immunoblot analysis showing the time course of EphA2 phosphorylation in oral epithelial cells that had been infected with yeast-phase C. albicans SC5314 for the indicated times. ( d ) EphA2 phosphorylation after 15-min infection with either C. albicans yeast or pregerminated hyphae. H, hyphae; Y, yeast. ( e ) Effects of C. albicans (SC5314, 529L, efg1/cph1 ), Candida glabrata , and Saccharomyces cerevisiae on EphA2 phosphorylation. Cg, C. glabrata ; Sc, S. cerevisiae . ( f–h ) Time course (in minutes) of EphA2 phosphorylation induced by zymosan ( f ), laminarin ( g ), and heat-killed C. albicans SC5314 (HK Ca ) ( h ). ( i ) Binding of recombinant EphA2 to immobilized ephrin A1, zymosan, laminarin, mannan, and BSA, as determined by ELISA. Box whisker plots show median and range of 3 experiments, each performed in triplicate. Statistical analysis of binding is shown relative to wells coated with BSA. EFNA1, ephrin A1; lam, laminarin; man, mannan; zym, zymosan ( j ) EphA2 (red) and Fc-dectin-1 (green) bind to exposed β-glucan on yeast-phase C. albicans . Results are representative of 3 independent experiments. Densitometric analyses of replicate immunoblots are shown in ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant (two-tailed Student’s t-test assuming unequal variances). Scale bars 5 μm.

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Infection, Expressing, Staining, Negative Control, Western Blot, Phospho-proteomics, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Whisker Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a–b ) Effects of EphA2 depletion with siRNA on the endocytosis of C. albicans by oral epithelial cells ( a ) and extent of C. albicans -induced host cell damage ( b ). ( c ) Effects of inhibition of EphA2 with dasatinib or an EphA2 antagonist and/or inhibition of the epidermal growth factor receptor (EGFR) with gefitinib on the endocytosis of C. albicans by oral epithelial cells. Box whisker plots show median and range of three experiments, each performed in triplicate. ( d ) Effect of EphA2 siRNA on EGFR phosphorylation at Y1068 in oral epithelial cells infected with C. albicans . ( e ) Effect of inhibition of EGFR with gefitinib on the phosphorylation of epithelial cell EphA2 in response to C. albicans infection. ( f ) Immunoblots showing the effects of EphA2 siRNA on the phosphorylation of epithelial cell MEK1/2 and c-FOS in response to C. albicans . ( g ) Effect of treatment of epithelial cells with an anti-dectin-1 mAb on the phosphorylation of p65 S536 induced by C. albicans. ( h ) Time course (in min) of MEK1/2 phosphorylation induced by zymosan. ( i ) Effect of EGFR inhibition on MEK1/2 phosphorylation in response to C. albicans . ( j ) Transient MEK1/2 activation in response to heat-killed C. albicans . Densitometric analyses of replicate immunoblots are shown in ).ANT, EphA2 antagonist; DAS, dasatinib; GEF, gefitinib; p.i., post-infection; HK Ca , Heat-killed C. albicans ; zym, zymosan. **** P < 0.0001 (two-tailed Student’s t-test assuming unequal variances).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Inhibition, Whisker Assay, Phospho-proteomics, Infection, Western Blot, Activation Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Immunoblot demonstrating that C. albicans infection induces phosphorylation of Stat3. ( b ) Effects of the EphA2 depletion with siRNA on C. albicans -induced phosphorylation of Stat3. ( c ) Time course of Stat3 phosphorylation induced by zymosan. Densitometric analyses of replicate immunoblots are shown in Supplementary Fig. 19 . ( d ) Oral epithelial cells were incubated with inhibitors of EphA2 and Stat3 and then infected with C. albicans for 5 h, after which chemokines and the S100a8 alarmin mRNA levels were determined by real-time PCR. Box whisker plots show median and range of 2 experiments, each performed in triplicate and are presented as fold induction relative to uninfected epithelial cells. ( e ) EphA2 depletion with siRNA reduces epithelial cell production of human β defensin 2, cytokines and chemokines in response to 8 h of C. albicans infection. Box whisker plots show median and range of 3 experiments, each performed in duplicate. ( f ) Regulation of the oral epithelial cell pro-inflammatory response to C. albicans by Stat3 and dectin-1. Box whisker plots show median and range of 3 experiments, each performed in duplicate. ANT, EphA2 antagonist; ctrl, control; DAS, dasatinib; hBD2, human β defensin 2; MOI, multiplicity of infection; Stat3 INH, Stat3 inhibitor; UNINF, uninfected. ** P < 0.01, *** P < 0.001, **** P < 0.0001; (two-tailed Student’s t-test assuming unequal variances).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Western Blot, Infection, Phospho-proteomics, Incubation, Real-time Polymerase Chain Reaction, Whisker Assay, Control, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Immunoblots showing the effects of increasing C. albicans inoculum on the extent of EphA2 phosphorylation. Densitometric analyses of replicate immunoblots are shown in . ( b ) Effects of C. albicans inoculum on epithelial cell secretion of IL-8 and hBD2. Box whisker plots show median and range of 3 independent experiments in duplicates. ctrl, control; hBD2, human β defensin 2; MOI, multiplicity of infection; Stat3 INH, Stat3 inhibitor; UNINF, uninfected. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; (two-tailed Student’s t-test assuming unequal variances).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Western Blot, Phospho-proteomics, Whisker Assay, Control, Infection, Two Tailed Test

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( a ) Oral fungal burden of immunocompetent wild-type and EphA2 −/− mice with oropharyngeal candidiasis after 1 d of infection. Results are median ± interquartile range of 4 mice per group in a single experiment. * P < 0.05 (Mann-Whitney Test) ( b ) Level of chemokines and cytokines in the tongue homogenates of immunocompetent wild-type and EphA2 −/− mice with OPC after 1 d of infection. Box whisker plots show median and range of 4 mice in each group, tested in duplicate in a single experiment. ( c ) Body weight wild-type and EphA2 −/− mice that were immunosuppressed with triamcinolone (7 mg/kg) prior to oral infection with C. albicans . Results are mean ± SD. * P < 0.05 (Holm-Sidak method). ( d ) Oral fungal burden of triamcinolone treated (7 mg/kg) wild-type and EphA2 −/− mice after 4 d of infection ( e–h ). Wild-type mice were immunosuppressed with triamcinolone (15mg/kg), treated with either DAS or the vehicle control, and orally inoculated with C. albicans . They were analyzed after 4 d of infection. ( e ) Oral fungal burden. Results are the median ± interquartile range of combined data from 2 independent experiments for a total of 14 mice in the control group and 13 mice in the DAS group. **** P < 0.0001 (Mann-Whitney Test) ( f ) Liver fungal burden. Results are median ± interquartile range of 7 mice per group in a single experiment. ** P < 0.01 (Mann-Whitney Test) ( g ) Immunohistochemistry of the tongue of a control mouse showing that β-glucan is expressed on the fungal cell surface during oropharyngeal candidiasis. The tongue was stained with Fc-Dectin-1 (green), an anti- Candida antibody (red), and DAPI (blue) Scale bar 100 μm. Results are representative of 3 mice from the same experiment. ( h ) mRNA expression of the indicated inflammatory mediators in the mouse tongue after 4 d of infection. mRNA levels were determined by ΔΔ CT method and normalized to GAPDH. Results are presented as fold change relative to the vehicle control mice. Data from two mice (M1 and M2) are presented. DAS, dasatinib; Veh, vehicle; WT, wild type.

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Infection, MANN-WHITNEY, Whisker Assay, Control, Immunohistochemistry, Staining, Expressing

Journal: Nature microbiology

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

doi: 10.1038/s41564-017-0059-5

Figure Lengend Snippet: ( Left panel ) EphA2 (blue) and dectin-1 (green) bind to exposed β-glucan on the fungal surface. Binding to EphA2 is independent of Ca 2+ and activates mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (Stat3). Binding to dectin-1 requires Ca 2+ and activates nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB). ( Right panel ) During fungal proliferation, prolonged activation of EphA2 via EGFR (grey) induces the endocytosis of C. albicans and a pro-inflammatory response in the epithelial cells. EphA2-EGFR induces endocytosis and triggers MAPK signaling. EphA2 also activates Stat3 Activation of the Stat3 and MAPK pathways leads to the release of alarmins, cytokines, chemokines, and host defense peptides (HDPs, orange helix).

Article Snippet: After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody.

Techniques: Binding Assay, Activation Assay