Journal: Cell

Article Title: Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization

doi: 10.1016/j.cell.2018.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Biotinylated hyaluronan binding protein (EMD Millipore, 1:50) was also used as a probe, and detected with the VECTASTAIN Elite ABC-HRP Kit (Vector Laboratories).

Techniques: Western Blot, Immunohistochemistry, Recombinant, Binding Assay, Transfection, Protein Extraction, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Luciferase, Colorimetric Assay, Activity Assay, Microarray, Generated, shRNA, CRISPR, Software, In Vivo Imaging, Microscopy

Journal: Cell

Article Title: Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization

doi: 10.1016/j.cell.2018.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Polybrene , Santa Cruz , Cat# sc-134220.

Techniques: Western Blot, Immunohistochemistry, Recombinant, Binding Assay, Transfection, Clinical Proteomics, Membrane, Protein Extraction, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Luciferase, cDNA Synthesis, Colorimetric Assay, Activity Assay, Microarray, Generated, Control, shRNA, CRISPR, Software, In Vivo Imaging, Microscopy

Journal: Cell

Article Title: Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization

doi: 10.1016/j.cell.2018.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Screening for activation (phosphorylation) of receptor tyrosine kinases was performed using the Proteome Profiler Human Phospho-RTK Array Kit (R&D Systems) per manufacturer’s protocol.

Techniques: Western Blot, Immunohistochemistry, Recombinant, Binding Assay, Transfection, Clinical Proteomics, Membrane, Protein Extraction, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Luciferase, cDNA Synthesis, Colorimetric Assay, Activity Assay, Microarray, Generated, Control, shRNA, CRISPR, Software, In Vivo Imaging, Microscopy

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Gateway-based cloning and expression system. (A) An entry clone containing the leader peptide and extracellular domain (ECD) of interest is recombined via the LR reaction with a destination vector to generate an expression clone. This incorporates the ECD fused to EGFP, an Avitag biotin acceptor peptide, a PP cleavage site, and a GPI signal sequence. (B) Following expression in mammalian cells, surface-presented protein can be cleaved with PP and biotinylated with BirA either at the cell surface or following cleavage. This versatile cloning vector enables multiple assays with either cell-expressed or soluble versions of the protein of interest.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Cloning, Expressing, Plasmid Preparation, Sequencing

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Expression of EGFP fusion proteins in CHO cells measured by flow cytometry. Cell surface expression of EGFP fusion proteins was measured on living cells using biotinylated anti-GFP followed by streptavidin–APC and detected on the FL-4 channel (emission maximum 660 nm). Total endogenous GFP was detected on the FL-1 channel (emission maximum 509 nm). CHO cells were either nontransfected (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), siglec-9 (S9), BACE, JAM-B, or JAM-C.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Expressing, Flow Cytometry

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Binding of polymeric glycan probes to siglec-expressing CHO cells. Either nontransfected control cells (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), or siglec-9 (S9) were incubated with biotinylated PAA probes carrying either lactose, Sia2,8Sia, SLe x , or 6′SU-SLe x and binding detected with streptavidin–APC. See for carbohydrate sequences.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Binding Assay, Glycoproteomics, Expressing, Control, Incubation

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Preparation of soluble siglecs and protein–glycan interactions measured on glycan arrays. (A) Western blot of soluble material released from siglec-expressing CHO cells either treated (+) or not treated (−) with PP. Supernatants containing cleaved siglec-7–EGFP (S7), siglec-8–EGFP (S8), and siglec-9–EGFP (S9) were probed with biotinylated goat anti-GFP followed by streptavidin–alkaline phosphatase. (B) Western blot of cleaved material either pretreated (+) or not pretreated (−) with BirA enzyme and probed with streptavidin–alkaline phosphatase. (C) Dose–response microarray analysis of the binding of siglecs to lipid-linked oligosaccharide probes. Microarrays of six oligosaccharide probes (structures are shown in ) were generated on nitrocellulose-coated glass slides. Each probe was printed in duplicate at 1 ( ), 1.33 ( ), 1.66 ( ), 2.33 ( ), 3.33 ( ), and 5 ( ) fmol/spot. Binding of biotinylated siglec-7–EGFP (top panel), siglec-8–EGFP (middle panel), and siglec-9–EGFP (bottom panel) chimeras was detected with AlexaFluor-647-labeled streptavidin as described in Materials and methods.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Glycoproteomics, Western Blot, Expressing, Microarray, Binding Assay, Generated, Labeling

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Measurement of protein–glycosaminoglycan interactions. Here 8mer saccharides derived from a partial heparinase I digestion of porcine mucosal heparin were spotted at different concentrations onto GAPS II microarray slides and incubated sequentially with 20 nM biotinylated protein–EGFP followed by AlexaFluor-546-labeled streptavidin. (A) Microarray image showing duplicate spots representative of 10 replicate spots. Contrast and brightness has been adjusted to show spot signal with respect to equivalent background signal. S9, siglec-9; −, streptavidin detection reagent applied to subarrays where no protein was incubated. (B) Graphs showing mean spot intensities against saccharide spotting concentration for different proteins. Spot intensity was normalized relative to background intensity by subtraction of the local background for each spot and the mean standard deviation of these values calculated for 10 replicate spots.

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Derivative Assay, Microarray, Incubation, Labeling, Concentration Assay, Standard Deviation

Journal: Analytical Biochemistry

Article Title: An expression system for screening of proteins for glycan and protein interactions

doi: 10.1016/j.ab.2010.12.036

Figure Lengend Snippet: Binding of biotinylated JAM-B–EGFP and JAM-C–EGFP chimeras to CHO cells expressing JAM-B and JAM-C. (A) Western blot of biotinylated JAM-C or JAM-B probed with streptavidin–alkaline phosphatase shows the presence of a single species for each protein at the expected molecular weight. (B) Wild-type CHO cells (black line) or CHO cells expressing JAM-B (red and green lines) were incubated with either biotinylated JAM-C–EGFP (black and green lines) or biotinylated JAM-B–EGFP (red line) at 1 μg/ml. Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (C) Wild-type CHO cells (black line) or CHO cells expressing JAM-C (red and green lines) were incubated with either biotinylated JAM-B–EGFP (black and green lines) or biotinylated JAM-C–EGFP (red line). Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The slides were rinsed with blocking buffer and overlaid for 1.5 h with the biotinylated siglec–GFP constructs that had been precomplexed for 1 h at 4 °C with AlexaFluor-647–streptavidin (Molecular Probes) (1:1, w/w) and rabbit anti-GFP (Invitrogen) (1:6, w/w).

Techniques: Binding Assay, Expressing, Western Blot, Molecular Weight, Incubation, Flow Cytometry

Journal: Cell

Article Title: Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization

doi: 10.1016/j.cell.2018.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For HABP staining, cell membranes remained intact and were blocked using an avidin/biotin blocking kit (Vector Laboratories).

Techniques: Western Blot, Immunohistochemistry, Recombinant, Binding Assay, Transfection, Protein Extraction, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Luciferase, Colorimetric Assay, Activity Assay, Microarray, Generated, shRNA, CRISPR, Software, In Vivo Imaging, Microscopy

Journal:

Article Title: Detection of Protein Biomarkers using RNA Aptamer Microarrays and Enzymatically Amplified SPR Imaging

doi: 10.1021/ac061849m

Figure Lengend Snippet: Summary of all DNA and RNA sequences used for the enzymatically amplified SPRI detection of hTh and VEGF. Fifteen thymine bases were attached to the 3′ end of the D 1 aptamer as a spacer to place the aptamer further away from the surface. Eight adenosine bases were added to the 3′ ends of RNA aptamers R 1 , R 2 and R V to facilitate the surface ligation reaction.

Article Snippet: Human thrombin (Haematologic Technologies Inc.), unmodified thrombin antibody and horseradish peroxidase conjugated thrombin antibody (Enzyme Research), recombinant human vascular endothelial growth factor (VEGF) (165-amino acid residues; Sigma-Aldrich), biotinylated anti-VEGF antibody (R&D Systems), anti-biotin horseradish peroxidase (Jackson ImmunoResearch), T4 RNA ligase (Epicentre Biotechnology) were all diluted to the desired concentration before use.

Techniques: Amplification, Ligation, Control

Journal:

Article Title: Detection of Protein Biomarkers using RNA Aptamer Microarrays and Enzymatically Amplified SPR Imaging

doi: 10.1021/ac061849m

Figure Lengend Snippet: Structures of the aptamers used in a four-component microarray for the identification of a thrombin aptamer to be used for thrombin detection in a surface sandwich assay. The four aptamer array elements are: R1, an RNA aptamer for human thrombin; D1, a DNA aptamer for human thrombin; R2, an RNA aptamer that was selected for bovine thrombin but can also bind to human thrombin; RV, an RNA aptamer for the human vascular endothelial growth factor (VEGF). The boxed guanine bases in the D1 aptamer form a G-quadruplex structure (not pictured) that is responsible for human thrombin binding. RV was used as a negative control in the thrombin detection. The pattern of the four-component microarray is shown at the top of this figure.

Article Snippet: Human thrombin (Haematologic Technologies Inc.), unmodified thrombin antibody and horseradish peroxidase conjugated thrombin antibody (Enzyme Research), recombinant human vascular endothelial growth factor (VEGF) (165-amino acid residues; Sigma-Aldrich), biotinylated anti-VEGF antibody (R&D Systems), anti-biotin horseradish peroxidase (Jackson ImmunoResearch), T4 RNA ligase (Epicentre Biotechnology) were all diluted to the desired concentration before use.

Techniques: Microarray, Binding Assay, Negative Control

Journal:

Article Title: Detection of Protein Biomarkers using RNA Aptamer Microarrays and Enzymatically Amplified SPR Imaging

doi: 10.1021/ac061849m

Figure Lengend Snippet: Enzymatically amplified SPRI detection of 500 fM hTh. (a) Schematic showing the surface R2 aptamer-hTh-HRP conjugated antibody sandwich structure. This was created by sequentially flowing hTh and the HRP conjugated monoclonal hTh antibody onto a surface immobilized R2 aptamer. (b) Pattern of the three-component RNA microarray composed of R2, RV, and RC for hTh detection. R2 is the RNA aptamer for hTh, RV is the RNA aptamer for VEGF, and RC is a control RNA sequence with no hairpin structures (see Table 1 for sequences). (c) An SPR difference image obtained from the detection of 500 fM hTh using the HRP enzymatic amplification reaction. Only R2 aptamer array elements showed a 1.6% increase in ΔR.

Article Snippet: Human thrombin (Haematologic Technologies Inc.), unmodified thrombin antibody and horseradish peroxidase conjugated thrombin antibody (Enzyme Research), recombinant human vascular endothelial growth factor (VEGF) (165-amino acid residues; Sigma-Aldrich), biotinylated anti-VEGF antibody (R&D Systems), anti-biotin horseradish peroxidase (Jackson ImmunoResearch), T4 RNA ligase (Epicentre Biotechnology) were all diluted to the desired concentration before use.

Techniques: Amplification, Microarray, Control, Sequencing

Journal:

Article Title: Detection of Protein Biomarkers using RNA Aptamer Microarrays and Enzymatically Amplified SPR Imaging

doi: 10.1021/ac061849m

Figure Lengend Snippet: Enzymatically amplified SPRI detection of 1 pM VEGF. (a) Schematic illustrating the surface sandwich structure formed by the sequential adsorption of VEGF (1 pM), a biotinylated VEGF antibody (10 nM), and an anti-biotin conjugated HRP (50 nM) onto VEGF aptamer array elements. (b) Pattern of the three-component RNA microarray consisting of R2, RV, and RC for VEGF detection. The RNA sequences are listed in Table 1. (c) An SPR difference image obtained from the detection of 1 pM VEGF with the HRP enzymatic amplification reaction. A ΔR increase of ~4.0% was observed at only the RV array elements.

Article Snippet: Human thrombin (Haematologic Technologies Inc.), unmodified thrombin antibody and horseradish peroxidase conjugated thrombin antibody (Enzyme Research), recombinant human vascular endothelial growth factor (VEGF) (165-amino acid residues; Sigma-Aldrich), biotinylated anti-VEGF antibody (R&D Systems), anti-biotin horseradish peroxidase (Jackson ImmunoResearch), T4 RNA ligase (Epicentre Biotechnology) were all diluted to the desired concentration before use.

Techniques: Amplification, Adsorption, Microarray

Journal: Cell

Article Title: Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization

doi: 10.1016/j.cell.2018.08.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SNL76/7 (mouse embryonic fibroblast) , ATCC , RRID: CVCL_K227.

Techniques: Western Blot, Immunohistochemistry, Recombinant, Binding Assay, Transfection, Clinical Proteomics, Membrane, Protein Extraction, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Luciferase, cDNA Synthesis, Colorimetric Assay, Activity Assay, Microarray, Generated, Control, shRNA, CRISPR, Software, In Vivo Imaging, Microscopy

Journal: The Journal of Neuroscience

Article Title: Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease

doi: 10.1523/JNEUROSCI.1327-16.2016

Figure Lengend Snippet: ADDLs reduce total and surface expression of EphB2, as well as surface expression of GluN2B-containing NMDA receptors in cultured hippocampal neurons. A, Effect of ADDLs on the expression of EphB2. Both the total and surface expressions of EphB2 were decreased. B, Effect of ADDLs on expression of the GluN2B receptor. The surface expression of GluN2B and the phosphorylated level of GluN2B at Y1472 were reduced, whereas the total expression of GluN2B was not changed. C, Effect of ADDLs on expression of the GluN1 receptor. The surface expression of GluN1 was reduced, but the total expression of GluN1 was not changed. D, Effect of ADDLs on expression of the GluN2A receptor. Neither the total or the surface expression of GluN2A was changed. E, ADDLs reduce surface expression of EphB2 with surface biotinylation assay in cultured hippocampal neurons. F, ADDLs reduce the surface expression of GluN2B with surface biotinylation assay in cultured hippocampal neurons. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control (Con). Data are presented as mean ± SEM.

Article Snippet: Membranes were blocked by 5% nonfat milk in TBST, then incubated at 4°C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz Biotechnology), GluN2B (1:1000; Santa Cruz Biotechnology), p-GluN2B (Y1472) (1:1000; Millipore), p-EphB2 (Y594) (1:1000; Abcam), β-actin (1:2000; Santa Cruz Biotechnology), and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology).

Techniques: Expressing, Cell Culture, Surface Biotinylation Assay

Journal: The Journal of Neuroscience

Article Title: Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease

doi: 10.1523/JNEUROSCI.1327-16.2016

Figure Lengend Snippet: Identification of the binding sites of the EphB2 FN domain on ADDLs. A, Prepared ADDLs coimmunoprecipitate with EphB2 from homogenates of the hippocampus. First, 500 μg of lysate was incubated with ADDLs (22.5 μg) overnight at 4°C with 4 μg of anti-β amyloid and 1–16 (6E10) monoclonal antibodies (Covance) added. Coimmunoprecipitation was performed according to the Catch and Release kit (Millipore) as described in the user's manual. Samples were subjected to SDS-PAGE followed by immunoblotting analysis using anti-EphB2 antibodies. B, Immunoblot results of the formaldehyde-mediated in vitro cross-linked products of ADDLs and FN. ADDLs (0.5 mg/ml) and FN (0.5 mg/ml) were mixed and incubated at RT for 0.5 h before the addition of formaldehyde. After the reaction was quenched by Tris-HCl (0.5 m), the cross-linked samples were subjected to SDS-PAGE and immunoblotting analysis. C, In vitro peptide scan of potential ADDL-interacting sites in the EphB2 FN domain. Overlapping 10-mer peptides with 3 aa shifts derived from the EphB2 FN domain were synthesized on cellulose membranes. Numbers denote the identity of the first and the last peptide of each line of the peptide array. The membrane was incubated with ADDLs and the bound protein was visualized immunologically using the β amyloid and 1–16 (6E10) monoclonal antibodies. Dark spots represent the peptides that had bound to ADDLs. Four spots as indicated by arrows were considered as potential binding regions. D, Peptide sequences of Pep21, Pep25, Pep32, and Pep63, respectively.

Article Snippet: Membranes were blocked by 5% nonfat milk in TBST, then incubated at 4°C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz Biotechnology), GluN2B (1:1000; Santa Cruz Biotechnology), p-GluN2B (Y1472) (1:1000; Millipore), p-EphB2 (Y594) (1:1000; Abcam), β-actin (1:2000; Santa Cruz Biotechnology), and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology).

Techniques: Binding Assay, Incubation, SDS Page, Western Blot, In Vitro, Derivative Assay, Synthesized, Peptide Microarray, Membrane

Journal: The Journal of Neuroscience

Article Title: Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease

doi: 10.1523/JNEUROSCI.1327-16.2016

Figure Lengend Snippet: Pep63 effectively interferes with the EphB2–ADDL interaction. A, Results of coimmunoprecipitation of ADDLs with EphB2 from hippocampus homogenates in the presence of Pep21, Pep25, Pep32, and Pep63, respectively. Pep32 and Pep63 were found to block the EphB2–ADDL interaction much more effectively than the other synthetic peptides, with the latter being slightly more effective than the former. B, Effect of Pep21, Pep25, Pep32, and Pep63 on the decreased surface expression of EphB2 induced by ADDLs in cultured hippocampal neurons. Treatment with Pep32 and Pep63 remarkably rescued the reduced surface expression of EphB2, with Pep63 displaying the best protective effect. C, Identification of the optimal neuroprotective concentration of Pep63 in cultured hippocampal neurons. Pep63 was found to rescue the decreased protein levels of EphB2 induced by ADDLs significantly at all concentrations tested; its maximal protective effect was found at a concentration of 2.5 μg/ml. D, Detection of the phosphorylated level of EphB2 at pY594 upon Pep63 treatment for 6 h. The pY594 of EphB2 in cells treated with Pep63 was comparable to that of control cells. E, Detection of the impacts of Pep63 on the activation of EphB2 by ephrinB1-Fc in cultured hippocampal neurons. F, Pep63 prevents the disruption of the EphB2–GluN1 interaction induced by ADDLs. EphB2 immunoprecipitation from cultured hippocampal neurons revealed that the dissociation of EphB2/GluN1 complexes upon ADDLs treatment was restored by exogenous Pep63. **p < 0.01, ***p < 0.001 compared with the control group (Con). #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the ADDL group. Data are presented as mean ± SEM.

Article Snippet: Membranes were blocked by 5% nonfat milk in TBST, then incubated at 4°C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz Biotechnology), GluN2B (1:1000; Santa Cruz Biotechnology), p-GluN2B (Y1472) (1:1000; Millipore), p-EphB2 (Y594) (1:1000; Abcam), β-actin (1:2000; Santa Cruz Biotechnology), and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology).

Techniques: Blocking Assay, Expressing, Cell Culture, Concentration Assay, Activation Assay, Disruption, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease

doi: 10.1523/JNEUROSCI.1327-16.2016

Figure Lengend Snippet: Pep63 rescues the decreased total and surface expression of EphB2 induced by ADDLs in cultured hippocampal neurons. A, Pep63 rescues the decreased total and surface expression of EphB2 induced by ADDLs. B, Pep63 rescues the decreased surface expression of GluN2B-containing NMDA receptors induced by ADDLs. The reduced phosphorylated GluN2B at pY1472 was improved as well. C, Double staining against N-terminal surface EphB2 (S-EphB2, red) and synaptophysin (Syn, green) showed that treatment with Pep63 (2.5 μg/ml) rescued both the decreased total surface (red) and synaptic (yellow) expression of EphB2 induced by ADDLs (500 nm, 6 h). No change in synaptophysin was observed. Scale bars, top three panels, 70 μm; bottom panel, 10 μm. D, Quantitative analysis of the total surface EphB2 area. E, Quantitative analysis of the synaptic EphB2 area. Error bars indicate SEM from at least three independent experiments with 25 cells imaged per experimental condition in each experiment. ***p < 0.001 compared with the control group (Con). #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the ADDL group. Data are presented as mean ± SEM.

Article Snippet: Membranes were blocked by 5% nonfat milk in TBST, then incubated at 4°C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz Biotechnology), GluN2B (1:1000; Santa Cruz Biotechnology), p-GluN2B (Y1472) (1:1000; Millipore), p-EphB2 (Y594) (1:1000; Abcam), β-actin (1:2000; Santa Cruz Biotechnology), and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology).

Techniques: Expressing, Cell Culture, Double Staining

Journal: The Journal of Neuroscience

Article Title: Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease

doi: 10.1523/JNEUROSCI.1327-16.2016

Figure Lengend Snippet: Pep63 rescues the decreased total and surface expression of EphB2 and the surface expression of GluN2B-containing NMDA receptors in the hippocampus of APP/PS1 transgenic mice. A, Pep63 significantly rescued the reduced total expression of EphB2 in APP/PS1 transgenic mice (APP). B, Pep63 significantly improved the surface expression of EphB2 in APP/PS1 transgenic mice. C, Pep63 strikingly rescued the reduced surface expression of GluN2B subunits in APP/PS1 transgenic mice. D, Pep63 significantly enhanced the phosphorylated levels of GluN2B at pY1472 in APP/PS1 transgenic mice. E, Pep63 remarkably rescued the reduced surface expression of GluN1 subunits in APP/PS1 transgenic mice. F, Surface expression of GluN2A subunits was not changed and Pep63 had no effect on their surface expression in APP/PS1 transgenic mice. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the WT vehicle group; #p < 0.05, ##p < 0.01 compared with the APP vehicle group. Data are presented as mean ± SEM.

Article Snippet: Membranes were blocked by 5% nonfat milk in TBST, then incubated at 4°C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz Biotechnology), GluN2B (1:1000; Santa Cruz Biotechnology), p-GluN2B (Y1472) (1:1000; Millipore), p-EphB2 (Y594) (1:1000; Abcam), β-actin (1:2000; Santa Cruz Biotechnology), and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology).

Techniques: Expressing, Transgenic Assay

Journal: General physiology and biophysics

Article Title: The expression of P-gp in leukemia cells is associated with cross-resistance to protein N-glycosylation inhibitor tunicamycin.

doi: 10.4149/gpb_2016039

Figure Lengend Snippet: Figure 6. Concanavalin A (ConA) and Sambucus nigra agglutinin (SNA) binding to crude membrane fractions (detected by lectin-based microarray assay) and wheat germ agglutinin (WGA) binding to the cell surface (detected by flow cytometry) of P- gp-negative and P-gp-positive leukemia cells. The data are expressed as the mean values ± S.E.M. from three independent measure ments. * p < 0.02 vs. control C, + p < 0.02 vs. control in the absence of TNM. S, L1210 cells; R, L1210/R; T, L1210/T cells; C, SKM- 1 and MOLM-13 cells; V, SKM-1/VCR and MOLM-13/VCR cells; L, SKM-1/LEN and MOLM-13/LEN cells.

Article Snippet: After washing, printed proteins were allowed to interact with the biotinylated lectins ConA, SNA and WGA (Vector, Burlingame, USA) at room temperature for 1 h. Biotinylated lectins at the concentration of 12.5 μg/ml in PBST were loaded into subarrays using a multiwell microarray mask.

Techniques: Binding Assay, Membrane, Microarray, Flow Cytometry, Control

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Specificity of brachyury in the distinction of chordoma from clear cell renal cell carcinoma and germ cell tumors: a study of 305 cases.

doi: 10.1038/modpathol.2010.196

Figure Lengend Snippet: Figure 1 (a) Hematoxylin and eosin (H&E), nested and cord-like growth typical of chordoma with intracytoplasmic vacuolization; (b) strong nuclear immunoreactivity for brachyury was seen in all 12 chordoma cases (highlighted in high power inset); (c) H&E, clear cell renal cell carcinoma showing the characteristic nested pattern with surrounding vascular septa; (d) no immunoreactivity for brachyury was seen in renal cell carcinoma (n ¼ 184); (e) H&E, classic seminoma with clear cytoplasm; (f) no immunoreactivity for brachyury was seen in any of the germ cell tumors (n ¼ 111).

Article Snippet: Immunohistochemical staining using anti-brachyury antibody (rabbit polyclonal (1:50); Santa Cruz Biotechnology, Santa Cruz, CA, USA; Ventana Benchmark XT (Tuscon, AZ, USA) autostainer with EDTA retrieval) was performed using standard avidin-biotin technique on 4-mm thick formalin-fixed, paraffin-embedded tissue microarray sections mounted on charged slides baked at 601C for 1 h. To address the potential staining heterogeneity bias of a tissue microarray study, 10 whole sections of seminomas with associated intratubular germ cell neoplasia unclassified were also stained for brachyury, with the diagnoses confirmed by immunostaining for OCT3/4 (anti-OCT3/4; goat polyclonal (1:400); Santa Cruz Biotechnology; Leica Bond Max (Bannockburn, IL, USA) autostainer with citrate retrieval).

Techniques: