Journal: Sleep & Breathing = Schlaf & Atmung

Article Title: Altered profile of circulating endothelial progenitor cells in obstructive sleep apnea

doi: 10.1007/s11325-012-0781-4

Figure Lengend Snippet: Association between sleep parameters, endothelial progenitor cells, and advanced glycation end-products

Article Snippet: Then, 100 μL of citrated blood was incubated with monoclonal antibodies against human kinase insert domain receptor (KDR) (1:10, R&D Biosystems; FITC-conjugated), CD133 (1:10, Miltenyi; PE-conjugated), CD34 (1:10, Becton Dickinson; APC-conjugated), and CD45 (1:10, Beckman Coulter; PC7-conjugated) for 30 min. After incubation, blood was lysed and fixed by OptiLyse® C solution (Beckman Coulter).

Techniques:

Journal: Sleep & Breathing = Schlaf & Atmung

Article Title: Altered profile of circulating endothelial progenitor cells in obstructive sleep apnea

doi: 10.1007/s11325-012-0781-4

Figure Lengend Snippet: Characteristics of recruited subjects

Article Snippet: Then, 100 μL of citrated blood was incubated with monoclonal antibodies against human kinase insert domain receptor (KDR) (1:10, R&D Biosystems; FITC-conjugated), CD133 (1:10, Miltenyi; PE-conjugated), CD34 (1:10, Becton Dickinson; APC-conjugated), and CD45 (1:10, Beckman Coulter; PC7-conjugated) for 30 min. After incubation, blood was lysed and fixed by OptiLyse® C solution (Beckman Coulter).

Techniques:

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Designation of the DNA vaccine.(a) B cell epitope scanning of human DKK1 was performed with the software DNASTAR 7.1. (b) The affinity of epitopes was measured by indirect ELISA. (c) The separated epitopes were immunized BALB/c mice and the immunogenicity of epitopes was measured by sandwich ELISA. (d) The separated epitopes were injected to BALB/c mice for seven days. TRAP staining was performed to identify the mature osteoclasts. Magnification: 200x; data are expressed as the mean ± SEM.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Software, Indirect ELISA, Immunopeptidomics, Sandwich ELISA, Injection, Staining

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Construction of the DNA vaccine. (a) The maps of recombinant DKK1 DNA vaccine. (b) The amino acid sequence of human DKK1. (c) The recombinant amino acid sequence of DKK1 DNA vaccine. Red line, 110–144aa; green line, 153–181aa; orange line, 182–216aa; blue line, 228–253aa; black line, signal peptide; box, PADRE.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Recombinant, Sequencing

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Expression and immunogenicity of the DNA vaccine. (a–c) Expression of the multiepitope protein in vitro and in vivo were determined in cell culture supernatants by ELISA, cell lysates by Western blotting, and the muscles of mice by immunohistochemical analysis. (d-e) The DNA vaccine induced a specific IgG antibody against human DKK1. The titer and the end-point titer of the specific antibody were tested by ELISA. Bars indicate 100 μ m. Data are expressed as the mean ± SEM, * P < 0.05, *** P < 0.001.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Expressing, Immunopeptidomics, In Vitro, In Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Muscles, Immunohistochemical staining

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques:

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Establishment of a sandwich ELISA for detecting L 59 LAP-DPs. a Schematic diagram of the ELISA for L 59 LAP-DPs. In the ELISA, L 59 LAP-DPs are first captured by coated L59 antibodies (Ab) and then sandwiched by biotin-conjugated anti-LAP antibodies (αLAP-Ab), which form a complex with strep-AP. For detection, an enzyme substrate is added and absorbance at 405 nm was measured. b , c Incubation time- and PLK concentration-dependent increases in the absorbance of the samples containing LAP incubated with PLK. The LAP (final 25 nM) was digested with 12.5 nM (open triangle), 25 nM (open reverse triangle), and 50 nM (open square) PLK, or without PLK (open circle) for 0–90 min, and then the samples were diluted by 1/50 and subjected to the L 59 LAP-DP ELISA ( b ). Also, 25 nM rhLAP was digested with 50 nM PLK (open square) or PLN (cross mark), or PLK in the presence of 5 μM camostat mesilate (open diamond) ( c ). All data were presented as mean ± SD from two different experiments ​*p-value <0.05, **p-value <0.01, ***p-value<0.001 obtained comparing to corresponding control values

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Correlation among L 59 LAP-DPs and active TGF-β in the culture medium, and intracellular signal transduction. a , c The ×9CAGA-Luc-transformed CCL64 cells were cultured in PLK-added CM derived from HEK293T cells overexpressing hLTGF-β1. After 6 h, the levels of active TGF-β1 and L 59 LAP-DPs were determined by respective ELISAs ( a ), and the extent of TGF-β signaling was measured by luciferase activity in CCL64 cells ( c ). b , d The scatterplots between the levels of L 59 LAP-DPs and active TGF-β shown in a ( b ) and between increases in L 59 LAP-DP levels and increases in luminescence from the values obtained compared to basal levels in the absence of PLK shown in c ( d ). A significant positive correlation was seen ( b ) * p-value <0.05, ***p-value <0.001 obtained comparing to 0

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Transformation Assay, Cell Culture, Derivative Assay, Luciferase, Activity Assay