Journal:

Article Title: Physiological levels of tumstatin, a fragment of collagen IV ?3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via ?V?3 integrin

doi:

Figure Lengend Snippet: A: Proliferation assay in β3 integrin−/− endothelial cells. MLEC were isolated from wild-type (β3+/+) and β3 integrin−/− (β3−/−) mice as previously described (Maeshima et al., 2002). Proliferation was assessed by [3H]-thymidine incorporation. All groups represent triplicate analyses. The results are shown as the mean ± SEM. ** indicates p < 0.01; compared to control cells.

Article Snippet: They were incubated with various primary antibodies, i.e., rat anti-mouse CD31 (Pharmingen), rabbit anti-von Willebrand Factor (vWF) (Dako), rat anti-β1 integrin (Pharmingen), and hamster anti-β3 integrin (Pharmingen) antibodies at room temperature for 2 hr.

Techniques: Proliferation Assay, Isolation

Journal:

Article Title: Physiological levels of tumstatin, a fragment of collagen IV ?3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via ?V?3 integrin

doi:

Figure Lengend Snippet: A: Immunohistochemical staining with anti-β3 integrin (β3) antibody and CD31 or vWF was performed to assess localization of β3 integrin in blood vessels. Anti-CD31 antibody was used for the tumor and skin tissues and anti-vWF antibody was used for the liver tissue. β3 integrin-positive blood vessels (merge) are indicated with arrows. Scale bar: 50 μm.

Article Snippet: They were incubated with various primary antibodies, i.e., rat anti-mouse CD31 (Pharmingen), rabbit anti-von Willebrand Factor (vWF) (Dako), rat anti-β1 integrin (Pharmingen), and hamster anti-β3 integrin (Pharmingen) antibodies at room temperature for 2 hr.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Physiological levels of tumstatin, a fragment of collagen IV ?3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via ?V?3 integrin

doi:

Figure Lengend Snippet: A: Immunohistochemical staining with anti-β3 integrin (β3) antibody and CD31 was performed to assess localization of β3 integrin in the tumor blood vessels with volumes of 0.1, 0.5 and 3.0 cm3 in wild-type mice. β3 integrin-positive blood vessels (merge) are indicated with arrows. Scale bar: 50 μm.

Article Snippet: They were incubated with various primary antibodies, i.e., rat anti-mouse CD31 (Pharmingen), rabbit anti-von Willebrand Factor (vWF) (Dako), rat anti-β1 integrin (Pharmingen), and hamster anti-β3 integrin (Pharmingen) antibodies at room temperature for 2 hr.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Type VIII Collagen Stimulates Smooth Muscle Cell Migration and Matrix Metalloproteinase Synthesis after Arterial Injury

doi:

Figure Lengend Snippet: Photomicrograph of medial SMCs incubated on coverslips coated with 10 μg/ml type VIII collagen, then stained with an antibody against vinculin to localize focal adhesions. A: 2-hour incubation. B: 4-hour incubation. C: 4-hour incubation in presence of normal mouse IgG. D: 4-hour incubation in the presence of anti-β1 integrin antibody. Original magnification, ×1000.

Article Snippet: SMCs were incubated with anti-rat integrin β1 antibody (clone HA2/5, Pharmingen) or anti-rat integrin β3 antibody (clone F11, Pharmingen) at a concentration of 20 μg/ml for 30 minutes at room temperature before plating SMCs in wells and performing migration assays in the presence of the blocking antibodies.

Techniques: Incubation, Staining

Journal:

Article Title: Type VIII Collagen Stimulates Smooth Muscle Cell Migration and Matrix Metalloproteinase Synthesis after Arterial Injury

doi:

Figure Lengend Snippet: Inhibition of attachment of human HF16 smooth muscle cells to type VIII collagen with integrin-blocking antibodies. Cells were preincubated with normal mouse IgG (NIgG), or anti-α1 integrin, anti-α2 integrin, or anti-β1 integrin antibody for 30 minutes before plating on wells precoated with 20 μg/ml type VIII collagen. Cells were allowed to adhere for 90 minutes, and then attachment was quantitated by toluidine blue staining as described in Materials and Methods. Values are mean ± SEM. *Value is significantly different from NIgG.

Article Snippet: SMCs were incubated with anti-rat integrin β1 antibody (clone HA2/5, Pharmingen) or anti-rat integrin β3 antibody (clone F11, Pharmingen) at a concentration of 20 μg/ml for 30 minutes at room temperature before plating SMCs in wells and performing migration assays in the presence of the blocking antibodies.

Techniques: Inhibition, Blocking Assay, Staining

Journal:

Article Title: Type VIII Collagen Stimulates Smooth Muscle Cell Migration and Matrix Metalloproteinase Synthesis after Arterial Injury

doi:

Figure Lengend Snippet: A: Migration of medial SMCs toward type VIII collagen (•) or type I collagen (○). Type VIII or type I collagen was dissolved in the bottom of a chemotaxis chamber at the indicated concentrations, and cells were plated on nylon filters with pores 8 μm in diameter. Migration was measured by counting cells that migrated to the bottom of the filter and was expressed as cells per field at ×200 magnification. Values are mean ± SEM. B: Inhibition of migration of medial SMCs toward 50 μg/ml type VIII collagen after treatment with integrin antibodies. Medial SMCs were preincubated with normal mouse IgG (NIgG), anti-β3 integrin antibody (anti-β3), or anti-β1 integrin antibody (anti-β1) for 30 minutes before plating. Values are mean ± SEM. *Value is significantly different from NIgG.

Article Snippet: SMCs were incubated with anti-rat integrin β1 antibody (clone HA2/5, Pharmingen) or anti-rat integrin β3 antibody (clone F11, Pharmingen) at a concentration of 20 μg/ml for 30 minutes at room temperature before plating SMCs in wells and performing migration assays in the presence of the blocking antibodies.

Techniques: Migration, Chemotaxis Assay, Inhibition

Journal:

Article Title: Disruption of the Mouse ?-Calpain Gene Reveals an Essential Role in Platelet Function

doi: 10.1128/MCB.21.6.2213-2220.2001

Figure Lengend Snippet: Proteolysis and tyrosine phosphorylation of platelet proteins. Washed platelets were activated by either calcium ionophore A23187 (1 μM) or thrombin (10 nM) at 37°C with stirring, and samples were taken out at indicated times for gel electrophoresis. (a) SDS-PAGE and Coomassie blue-stained 6% gel of total platelet lysate (40 μl). Calcium ionophore A23187 was used as an agonist. Arrows indicate the positions of intact filamin and talin. The 190-kDa fragment of talin (solid arrowhead) and the 130- and 93-kDa cleavage products of filamin (open arrowheads) are shown. (b) SDS-PAGE and Coomassie blue-stained 6% gel of total platelet lysate (40 μl). Thrombin was used as an agonist. Other symbols are the same as shown in the previous panel. (c and d) Western blot analysis of total platelet protein samples shown in panels a and b. (c) Calcium ionophore A23187 treatment. (d) Thrombin-treated platelets. An equal amount of platelet lysate, normalized by protein concentration, was analyzed by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed with an antiphosphotyrosine monoclonal antibody (4G10). Note that the asterisk at p110 indicates the position of the β3 subunit of αIIbβ3 integrin. The same blots were stripped and reprobed to examine the proteolysis of β3 integrin and talin using defined antibodies. The bottom three panels show the results of Western blots using antibodies against β3 integrin and talin. The anti-Nβ3 antibody is specific for the N-terminal region of β3 integrin and was used to determine the total amount of β3 integrin in each lane. The anti-β3C antibody is cleavage sensitive and detects only the intact C terminus of β3 integrin (10). The anti-talin 8d4 antibody (Sigma) recognized the intact talin and cleaved the 190-kDa fragment but not the 50-kDa fragment. The same results were obtained in four independent experiments.

Article Snippet: Immunoprecipitations were performed using biotin-conjugated anti-integrin β3 antibody (Becton Dickinson) for 12 h at 4°C, followed by incubation with streptavidin-conjugated agarose beads (Pierce) for 2 h at 4°C.

Techniques: Nucleic Acid Electrophoresis, SDS Page, Staining, Western Blot, Protein Concentration

Journal:

Article Title: Disruption of the Mouse ?-Calpain Gene Reveals an Essential Role in Platelet Function

doi: 10.1128/MCB.21.6.2213-2220.2001

Figure Lengend Snippet: Immunoprecipitation and tyrosine phosphorylation of the β3 subunit of αIIbβ3 integrin. The β3 integrin was immunoprecipitated using biotin-conjugated anti-mouse antibody against the integrin β3 chain. Immunoprecipitates (IP) were analyzed by SDS-PAGE (8% gel), transferred to a nitrocellulose membrane, and blotted (IB) an antiphosphotyrosine antibody, 4G10 (upper panel). The same blot was stripped and blotted with an anti-Nβ3 antibody to normalize the amount of β3 integrin in each lane (lower panel).

Article Snippet: Immunoprecipitations were performed using biotin-conjugated anti-integrin β3 antibody (Becton Dickinson) for 12 h at 4°C, followed by incubation with streptavidin-conjugated agarose beads (Pierce) for 2 h at 4°C.

Techniques: Immunoprecipitation, SDS Page

Journal: Scientific Reports

Article Title: TRAF3 negatively regulates platelet activation and thrombosis

doi: 10.1038/s41598-017-17189-1

Figure Lengend Snippet: Platelet secretion and aggregation are defective in the TRAF3 knockout mice. ( a ) Washed platelets from TRAF3 knockout mice (TRAF3 −/− ) or wild-type littermates (TRAF3 +/+ ) were stimulated with various concentrations of thrombin or collagen and simultaneously recorded for ATP secretion and aggregation. The aggregation and ATP release traces are representatives of at least three different experiments. ( b ) Aggregation and ATP secretion results in the experiments described in A were quantitated. ( c ) Total serotonin in 1 × 10 9 TRAF3 +/+ and TRAF3 −/− platelets from four mice with each genotype was measured by O-phthalaldehyde assay as described under “Experimental Procedures” (n = 4). ( d ) Detection of P-selectin in platelet lysates from TRAF3 +/+ and TRAF3 −/− mice by Western blot (n = 4).

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse P-selectin and integrin β3 antibodies were from BD Pharmingen.

Techniques: Knock-Out, Western Blot

Journal:

Article Title: Clearance of Apoptotic Photoreceptors

doi:

Figure Lengend Snippet: Cell Counts per 1-mm Width of the Retina

Article Snippet: Blockade of Phagocytotic Process by Inhibiting Receptor Systems To investigate the participation of candidate receptors for phagocytosis of apoptotic photoreceptors, we used RGD peptide (Bachem, Bubendorf, Switzerland), anti-integrin β3 antibody (clone F11; BD Pharmingen, San Diego, CA) for integrin αvβ3, and anti-PSR antibody (Cascade BioScience, Winchester, MA).

Techniques:

Journal:

Article Title: Clearance of Apoptotic Photoreceptors

doi:

Figure Lengend Snippet: The blockade of the macrophage phagocytosis by inhibiting receptor systems. Fluorescent images (A–C) show apoptotic debris (TUNEL, green), nucleus (propidium iodide, red), and phase contrast images (gray). Scanning electron microscopy revealed eliminated apoptotic photoreceptor debris (arrows) and infiltrating macrophages (D–F). The infiltrating macrophages selectively recognize and phagocytose a large amount of TUNEL-positive apoptotic debris (A, green). In contrast, the function-blocking anti-PSR antibody blocks apoptotic debris recognition by infiltrating macrophages (B and E). The function-blocking peptide with integrin αvβ3-binding motif RGD does not block the recognition and binding of apoptotic debris by macrophages, but blocks internalization of these particles (C, green; and F). The binding of numerous apoptotic bodies to a single macrophage is shown (F, arrowheads). Original magnifications: ×400 (A–C); ×2500 (D–F). Scale bars, 5 μm.

Article Snippet: Blockade of Phagocytotic Process by Inhibiting Receptor Systems To investigate the participation of candidate receptors for phagocytosis of apoptotic photoreceptors, we used RGD peptide (Bachem, Bubendorf, Switzerland), anti-integrin β3 antibody (clone F11; BD Pharmingen, San Diego, CA) for integrin αvβ3, and anti-PSR antibody (Cascade BioScience, Winchester, MA).

Techniques: TUNEL Assay, Electron Microscopy, Blocking Assay, Binding Assay

Journal: Journal of Translational Medicine

Article Title: Integrins β1 and β3 are biomarkers of uterine condition for embryo transfer

doi: 10.1186/s12967-016-1052-0

Figure Lengend Snippet: Expression levels of integrins β1 and β3 in endometrial cells were negatively associated with E 2 levels in clinical samples. a , b Quantitative analysis of integrin β1 and β3 mRNA expression levels in the over physiological E2 group (OP). c , d Quantitative analysis of integrin β1 and β3 mRNA expression levels in the physiological effect group (NE). Double asterisk indicates significantly different from the control group, P < 0.01; NS not significantly different

Article Snippet: FITC labeled anti-integrin β3 antibody (CAT. 555753) was from BD Bioscience (San Jose, CA, USA).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Integrins β1 and β3 are biomarkers of uterine condition for embryo transfer

doi: 10.1186/s12967-016-1052-0

Figure Lengend Snippet: E 2 enhanced the expression and distribution of integrin β3 on the plasma membranes of Ishikawa cells. a Representative image of integrin β1 expression as detected by western blotting. b Quantitative analysis integrin β1 expression as detected by western blotting. c Quantitative analysis of integrin β1 expression on the plasma membranes of Ishikawa cells as detected by flow cytometry. d Representative image of integrin β3 expression as detected by western blotting. e Quantitative analysis integrin β3 expression as detected by western blotting. f Quantitative analysis of integrin β3 distribution on the plasma membranes of Ishikawa cells as detected by flow cytometry. Double asterisk indicates significantly different from the control group, P < 0.01

Article Snippet: FITC labeled anti-integrin β3 antibody (CAT. 555753) was from BD Bioscience (San Jose, CA, USA).

Techniques: Expressing, Western Blot, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Integrins β1 and β3 are biomarkers of uterine condition for embryo transfer

doi: 10.1186/s12967-016-1052-0

Figure Lengend Snippet: P4 enhanced the expression of integrin β1 and its distribution on the plasma membranes of Ishikawa cells. a Representative image of integrin β1 expression as detected by western blotting. b Quantitative analysis of integrin β1 expression as detected by western blotting. c Quantitative analysis of integrin β1 distribution on the plasma membranes of Ishikawa cells as detected by flow cytometry. d Representative image of integrin β3 expression as detected by western blotting. e Quantitative analysis of integrin β3 expression as detected by western blotting. f Quantitative analysis of integrin β3 distribution on the plasma membranes of Ishikawa cells as detected by flow cytometry. Double asterisk indicates significantly different from the control group, P < 0.01

Article Snippet: FITC labeled anti-integrin β3 antibody (CAT. 555753) was from BD Bioscience (San Jose, CA, USA).

Techniques: Expressing, Western Blot, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Integrins β1 and β3 are biomarkers of uterine condition for embryo transfer

doi: 10.1186/s12967-016-1052-0

Figure Lengend Snippet: P4 eliminated E2-induced reductions in the expression and plasma membrane distribution of integrin β3 in Ishikawa cells. a Representative image of integrin β1 expression as detected by western blotting. b Quantitative analysis of integrin β1 expression as detected by western blotting. c Quantitative analysis of integrin β1 expression on the plasma membranes of Ishikawa cells as detected by flow cytometry. d Representative image of integrin β3 expression as detected by western blotting. e Quantitative analysis of integrin β3 expression as detected by western blotting. f Quantitative analysis of integrin β3 distribution on the plasma membranes of Ishikawa cells as detected by flow cytometry. Double asterisk indicates significantly different in three separate assays, P < 0.01

Article Snippet: FITC labeled anti-integrin β3 antibody (CAT. 555753) was from BD Bioscience (San Jose, CA, USA).

Techniques: Expressing, Western Blot, Flow Cytometry

Journal: Oncology Letters

Article Title: Integrin αVβ 3 -targeted SPECT/CT for the assessment of Bevacizumab therapy in orthotopic lung cancer xenografts

doi: 10.3892/ol.2018.7901

Figure Lengend Snippet: Images of A549 and PC-3 tumor tissues following immunohistochemical staining for integrin β 3 and CD31 in vehicle- and bevacizumab-treated groups to illustrate the alterations in integrin β 3 expression levels and blood vessel density during therapy. Tumor tissues were harvested at −1, 5 and 15 days after initiation of bevacizumab therapy. CD31 was used as the marker for tumor blood vessels (mature and newly formed), which was visualized with fluorescein isothiocyanate (green color). Integrin β 3 was visualized with cyanine 3 (red color). The yellow color in overlay images indicates the integrin β 3 expression on new blood vessels. Original magnification ×200. CD31, cluster of differentiation 31.

Article Snippet: The sections were then blocked with 10% goat serum (Abcam, Cambridge, MA, USA) for 30 min at room temperature and then incubated with rat anti-integrin β 3 antibody (1:100; cat. no. 181720; BD Biosciences, Franklin Lakes, NJ, USA) and rat anti-CD31 antibody (1:100; cat. no. 551262; BD Biosciences) for 1 h at room temperature.

Techniques: Immunohistochemical staining, Staining, Expressing, Marker

Journal: Bio-protocol

Article Title: Measurement of redox states of the β3 integrin disulfide bonds by Mass Spectrometry

doi: 10.21769/BioProtoc.3156

Figure Lengend Snippet: Monoisotopic m/z ratios for the β3 integrin YCNCTTR peptide alkylated with 12 C-IPA and 13 C-IPA

Article Snippet: Ziptip ® C18 pipette tip (Millipore, catalog number: ZTC18S96) 1.5 ml tubes Natural human αIIbβ3 protein purified from human platelets (Abcam, catalog number: ab95130) Anti-integrin β3 (GPIIIa, CD61) PSI domain [AP-3] antibody (Kerafast, catalog number: EBW106) 2-iodo-N-phenylacetamide ( 12 C-IPA) (Cambridge isotopes, catalog number: ULM-8131-PK) 13 C-2-iodo-N-phenylacetamide ( 13 C-IPA) (Cambridge isotopes, catalog number: CLM-8130-0.1) Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418) NuPAGE TM 4x LDS sample loading buffer (Thermo Fisher Scientific, catalog number: NP0007) NuPAGE TM 4-12% Bis-Tris gel 1.5 mm, 10-well (Thermo Fisher Scientific, catalog number: NP0335BOX) NuPAGE TM MOPS running buffer 20x (Thermo Fisher Scientific, catalog number: NP0001) Brilliant Blue G solution (Sigma, catalog number: B8522-1EA) Ammonium bicarbonate (Sigma, catalog number: A6141) Dithiothreitol (DTT) (Sigma, catalog number: D0632) Formic acid LC-MS grade (Thermo Fisher Scientific, catalog number: 28905) Trifluoroacetic acid (Sigma, catalog number: 302031) Formic acid (Sigma, catalog number: F0507) Acetonitrile HPLC grade (Fisher Chemical, catalog number: 10407440) PNGase F Proteomics grade enzyme (Sigma, catalog number: P7367) Sequencing grade bovine chymotrypsin (Roche, catalog number: 11418467001) Trypsin Gold, Mass spectrometry grade (Promega, catalog number: V5280) HCl Acetic acid Calcium chloride Methanol Coomassie blue NaCl KCl Na 2 HPO 4 KH 2 PO 4 PBS (see Recipes) Gel destaining solution (see Recipes) 50 mM 12 C-IPA/DMSO solution (see Recipes) 50 mM 13 C-IPA/DMSO solution (see Recipes) 5 mM 13 C-IPA/25 mM ammonium bicarbonate (see Recipes) 25 mM ammonium bicarbonate buffer (see Recipes) 25 mM ammonium bicarbondate/50% acetonitrile buffer (see Recipes) 25 mM ammonium bicarbonate/10 mM calcium chloride buffer (see Recipes) 1 M calcium chloride solution (see Recipes) 40 mM DTT/25 mM ammonium bicarbonate (see Recipes) 1 mM HCl solution (see Recipes) 50 mM acetic acid (see Recipes) 5% formic acid (see Recipes) 5% formic acid/50% acetonitrile buffer (see Recipes) 1% trifluoroacetic acid solution (see Recipes) Wetting solution (see Recipes) Equilibration solution (see Recipes) 0.1% formic acid (see Recipes) HPLC Buffer A (see Recipes) HPLC Buffer B (see Recipes)

Techniques: Sequencing