heparitinases Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    Millipore heparitinase
    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of <t>heparitinase</t> treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P
    Heparitinase, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/Millipore
    Average 96 stars, based on 121 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    86
    Seikagaku heparitinases
    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of <t>heparitinase</t> treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P
    Heparitinases, supplied by Seikagaku, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinases/product/Seikagaku
    Average 86 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    heparitinases - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    91
    Seikagaku heparitinase
    Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line. DRMs were prepared from confluent PTr cells as described in Materials and Methods . Collected fractions, were concentrated, treated with <t>heparitinase</t> I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).
    Heparitinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/Seikagaku
    Average 91 stars, based on 284 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    AMS Biotechnology heparitinase
    Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase <t>ABC/heparitinase</t> digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.
    Heparitinase, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/AMS Biotechnology
    Average 91 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    Associates of Cape Cod Inc heparitinase
    Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase <t>ABC/heparitinase</t> digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.
    Heparitinase, supplied by Associates of Cape Cod Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/Associates of Cape Cod Inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    85
    Seikagaku heparitinase enzyme
    Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without <t>heparitinase</t> treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western
    Heparitinase Enzyme, supplied by Seikagaku, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase enzyme/product/Seikagaku
    Average 85 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    heparitinase enzyme - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    89
    ICN Biomedicals heparitinase i
    The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of <t>heparitinase</t> treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.
    Heparitinase I, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 89/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase i/product/ICN Biomedicals
    Average 89 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    heparitinase i - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    85
    Millipore heparitinases
    The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of <t>heparitinase</t> treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.
    Heparitinases, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinases/product/Millipore
    Average 85 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    heparitinases - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Seikagaku heparitinase mixture
    The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of <t>heparitinase</t> treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.
    Heparitinase Mixture, supplied by Seikagaku, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase mixture/product/Seikagaku
    Average 85 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    heparitinase mixture - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    91
    IBEX Technologies heparitinase
    HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with <t>heparitinase</t> to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.
    Heparitinase, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/IBEX Technologies
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    BioMarin heparitinase
    <t>Heparitinase</t> treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p
    Heparitinase, supplied by BioMarin, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/BioMarin
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    85
    Millipore proteinase free heparitinase i
    TG2 extracellular location depends on the HS chains of HSPGs. Kidney cryosections were treated with 50 mU/ml protease-free <t>heparitinase</t> I (Hep-I) for 2 hours at 37°C. Extracellular TG2 was immunolabeled using mouse anti-TG2 IA12 antibody followed
    Proteinase Free Heparitinase I, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase free heparitinase i/product/Millipore
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    proteinase free heparitinase i - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of heparitinase treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P

    Journal: JCI Insight

    Article Title: Osteoblastic heparan sulfate regulates osteoprotegerin function and bone mass

    doi: 10.1172/jci.insight.89624

    Figure Lengend Snippet: Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of heparitinase treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P

    Article Snippet: Digestion of cell surface HS was performed by incubating live cells with 5 mIU/ml heparitinase (heparinase III) (MilliporeSigma, H8891) for 2 hours at 37°C.

    Techniques: Functional Assay, Generated, Affinity Chromatography, Binding Assay, Labeling, Staining, Marker, Activity Assay, Inhibition

    SB105_A10 binds to cell surface heparan sulfate. Vero cell monolayers were left untreated or treated with heparinase I or heparitinase I (2.5 U/ml) for 1 h at 37°C, washed with medium, and chilled at 4°C. Next, SB105_A10-FAM (5 μM)

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers ▿Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers ▿ †

    doi: 10.1128/AAC.00149-11

    Figure Lengend Snippet: SB105_A10 binds to cell surface heparan sulfate. Vero cell monolayers were left untreated or treated with heparinase I or heparitinase I (2.5 U/ml) for 1 h at 37°C, washed with medium, and chilled at 4°C. Next, SB105_A10-FAM (5 μM)

    Article Snippet: Vero cell monolayers were pretreated with 2.5 U of heparinase I (heparin lyase I) or heparitinase I (heparin lyase III) (Sigma) in PBS containing 1 mM CaCl2 , 1.5 mM MgCl2 , 0.1% glucose, and 0.1% bovine serum albumin or with buffer alone for 1 h at 37°C as described previously ( ).

    Techniques:

    Sulphated heparan sulphates are required for tau hyperphosphorylation in two cell models of tauopathy . Tau hyperphosphorylated at Ser199, Thr231, Ser262, and Ser396, total tau (Tau-5), and β-actin levels in cells treated with NaClO 3 in ( A ) SH/ox cells, and in ( B ) SH/hTau P301L cells. ( C ) Hyperphosphorylated tau at Ser199 and Ser396 in SH/hTau P301L cells treated or not with heparitinases I, II, and III cocktail (Hep-ase) or chondroitinase ABC (Cse). ( D ) Tau hyperphosphorylated at Ser199 in SH/ox cells after a stress pulse treated or not with heparitinase, Cse or NaClO 3 . ( E ) Tau hyperphosphorylated at Ser396 in SH/ox cells treated or not with heparitinase prior to the oxidative stress pulse. Data represent the mean of three different experiments performed in duplicate each time; error bars show SEM. ( F ) Western blots showing the phosphorylation of NF-kB p65 and p38 proteins in SH/ox ( left ) and in SH/hTau P301L ( right ) cells treated or not with the heparitinases cocktail or with NaClO 3 . Total levels of each protein, as well as that of β-actin, were used as loading controls. ( G ) ELISA assay showing the binding of heparin to tau (filled circles, solid line), Tau P301L (open circles, dashed line), GSK3B (upward triangles, spotted line), PP2A (downward triangles, spotted line), and PKA (diamonds, solid line). Data in histograms were made from the average signal obtained from the densitometric analysis of duplicates carried in each individual western blot, or by the average of two different western blots when duplicates were not performed.

    Journal: Brain

    Article Title: HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer’s disease-related tau pathology

    doi: 10.1093/brain/awv056

    Figure Lengend Snippet: Sulphated heparan sulphates are required for tau hyperphosphorylation in two cell models of tauopathy . Tau hyperphosphorylated at Ser199, Thr231, Ser262, and Ser396, total tau (Tau-5), and β-actin levels in cells treated with NaClO 3 in ( A ) SH/ox cells, and in ( B ) SH/hTau P301L cells. ( C ) Hyperphosphorylated tau at Ser199 and Ser396 in SH/hTau P301L cells treated or not with heparitinases I, II, and III cocktail (Hep-ase) or chondroitinase ABC (Cse). ( D ) Tau hyperphosphorylated at Ser199 in SH/ox cells after a stress pulse treated or not with heparitinase, Cse or NaClO 3 . ( E ) Tau hyperphosphorylated at Ser396 in SH/ox cells treated or not with heparitinase prior to the oxidative stress pulse. Data represent the mean of three different experiments performed in duplicate each time; error bars show SEM. ( F ) Western blots showing the phosphorylation of NF-kB p65 and p38 proteins in SH/ox ( left ) and in SH/hTau P301L ( right ) cells treated or not with the heparitinases cocktail or with NaClO 3 . Total levels of each protein, as well as that of β-actin, were used as loading controls. ( G ) ELISA assay showing the binding of heparin to tau (filled circles, solid line), Tau P301L (open circles, dashed line), GSK3B (upward triangles, spotted line), PP2A (downward triangles, spotted line), and PKA (diamonds, solid line). Data in histograms were made from the average signal obtained from the densitometric analysis of duplicates carried in each individual western blot, or by the average of two different western blots when duplicates were not performed.

    Article Snippet: For enzymatic treatments, differentiated cells were treated either with a heparitinase mix containing heparitinase I (2 U/ml), heparitinase II (0.2 U/ml), and heparitinase III (0.2 U/ml) (all from Sigma), or with chondroitinase ABC (0.1 U/ml) (Sigma) and incubated at 37°C for 1.5 or 6 h (as indicated).

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay

    3- O -sulphated heparan sulphates interact with tau under pathological conditions . Heparitinase (Hep-ase) treatment decreases membrane-associated and intracellular 3- O -sulphated heparan sulphates (red) staining and co-localization with tau. Z -projections are depicted for ( A ) control cells; ( B ) SH/ox cells treated with heparitinase before the oxidative stress pulse; and ( C ) SH/hTau P301L cells. ( D ) Quantification of heparan sulphates (HS) intracellular inclusions in SH/hTau P301L cells treated with hep-ase during 1.5 or 6 h. ( E ) FRET-based measurement of the molecular interaction between 3- O -sulphated heparan sulphates and tau in SH/ox cell lysates at different time points after the stress pulse. Calculated delta F (ΔF) shows a time-dependent molecular interaction abolished when cells were cultured in the presence of heparitinase I, II, III cocktail (Hep-ase). ( F ) ELISA competition assay showing the higher capacity of heparin, used as a prototype of 3- O -sulphate heparan sulphates, to inhibit the binding of tau to immobilized heparin compared to heparan sulphates. Data represents the mean of three different experiments performed in triplicate each time; error bars show SEM.

    Journal: Brain

    Article Title: HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer’s disease-related tau pathology

    doi: 10.1093/brain/awv056

    Figure Lengend Snippet: 3- O -sulphated heparan sulphates interact with tau under pathological conditions . Heparitinase (Hep-ase) treatment decreases membrane-associated and intracellular 3- O -sulphated heparan sulphates (red) staining and co-localization with tau. Z -projections are depicted for ( A ) control cells; ( B ) SH/ox cells treated with heparitinase before the oxidative stress pulse; and ( C ) SH/hTau P301L cells. ( D ) Quantification of heparan sulphates (HS) intracellular inclusions in SH/hTau P301L cells treated with hep-ase during 1.5 or 6 h. ( E ) FRET-based measurement of the molecular interaction between 3- O -sulphated heparan sulphates and tau in SH/ox cell lysates at different time points after the stress pulse. Calculated delta F (ΔF) shows a time-dependent molecular interaction abolished when cells were cultured in the presence of heparitinase I, II, III cocktail (Hep-ase). ( F ) ELISA competition assay showing the higher capacity of heparin, used as a prototype of 3- O -sulphate heparan sulphates, to inhibit the binding of tau to immobilized heparin compared to heparan sulphates. Data represents the mean of three different experiments performed in triplicate each time; error bars show SEM.

    Article Snippet: For enzymatic treatments, differentiated cells were treated either with a heparitinase mix containing heparitinase I (2 U/ml), heparitinase II (0.2 U/ml), and heparitinase III (0.2 U/ml) (all from Sigma), or with chondroitinase ABC (0.1 U/ml) (Sigma) and incubated at 37°C for 1.5 or 6 h (as indicated).

    Techniques: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Competitive Binding Assay, Binding Assay

    Neuropilin expression and glycosylation in human VSMCs and endothelial cells ( A ) Whole-cell lysates of HUVECs, HCAECs, HCASMCs and HAoSMCs were immunoblotted for NRP1, NRP2, VEGFR2, synectin, PDGFRα, PDGFRβ and GAPDH. NRP1 bands of approximately 130 kDa and > 250 kDa are indicated. ( B ) Cell-surface expression of NRP1 was examined in HUVECs and HCASMCs using flow cytometry as described in the Supplementary Online Data at http://www.BiochemJ.org/bj/435/bj4350609add.htm . KDR, kinase insert domain-containing receptor (VEGFR2); PE, phycoerythrin. ( C ) Confluent cultures of the cells indicated were pre-treated with (+) or without (−) 5 μg/ml tunicamycin for 16 h. Lysates were then prepared and immunoblotted with an antibody against NRP1 or NRP2. ( D ) Top and middle: HCASMCs were treated with chondroitinase, heparitinase or both enzymes combined (each at 1 unit/ml) for 4 h, and lysates were prepared and immunoblotted with an antibody against NRP1 or GAPDH. Bottom: NRP1 immunoprecipitates prepared from HCASMCs were incubated for 4 h with chondroitinase or heparitinase, and then immunoblotted with anti-NRP1 antibody. WB, Western blot; IP, immunoprecipitate. Results shown in ( A )–( D ) are representative of at least three independent experiments. Molecular masses are indicated in kDa. ( E ) Amounts of GAG-modified NRP1 after chondroitinase or heparitinase treatments in the Western blots shown in ( D ) were quantified by scanning densitometry, and used to calculate the relative levels of unmodified NRP1, HS-GAG–NRP1 and CS-GAG–NRP1 in HCASMCs. Results are mean percentages of total NRP1 immunoreactivity (non-GAG-modified 130 kDa NRP1 plus GAG-modified > 250 kDa NRP1).

    Journal: Biochemical Journal

    Article Title: Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

    doi: 10.1042/BJ20100580

    Figure Lengend Snippet: Neuropilin expression and glycosylation in human VSMCs and endothelial cells ( A ) Whole-cell lysates of HUVECs, HCAECs, HCASMCs and HAoSMCs were immunoblotted for NRP1, NRP2, VEGFR2, synectin, PDGFRα, PDGFRβ and GAPDH. NRP1 bands of approximately 130 kDa and > 250 kDa are indicated. ( B ) Cell-surface expression of NRP1 was examined in HUVECs and HCASMCs using flow cytometry as described in the Supplementary Online Data at http://www.BiochemJ.org/bj/435/bj4350609add.htm . KDR, kinase insert domain-containing receptor (VEGFR2); PE, phycoerythrin. ( C ) Confluent cultures of the cells indicated were pre-treated with (+) or without (−) 5 μg/ml tunicamycin for 16 h. Lysates were then prepared and immunoblotted with an antibody against NRP1 or NRP2. ( D ) Top and middle: HCASMCs were treated with chondroitinase, heparitinase or both enzymes combined (each at 1 unit/ml) for 4 h, and lysates were prepared and immunoblotted with an antibody against NRP1 or GAPDH. Bottom: NRP1 immunoprecipitates prepared from HCASMCs were incubated for 4 h with chondroitinase or heparitinase, and then immunoblotted with anti-NRP1 antibody. WB, Western blot; IP, immunoprecipitate. Results shown in ( A )–( D ) are representative of at least three independent experiments. Molecular masses are indicated in kDa. ( E ) Amounts of GAG-modified NRP1 after chondroitinase or heparitinase treatments in the Western blots shown in ( D ) were quantified by scanning densitometry, and used to calculate the relative levels of unmodified NRP1, HS-GAG–NRP1 and CS-GAG–NRP1 in HCASMCs. Results are mean percentages of total NRP1 immunoreactivity (non-GAG-modified 130 kDa NRP1 plus GAG-modified > 250 kDa NRP1).

    Article Snippet: Chondroitinase ABC and heparinase III (heparitinase) were from Sigma, and the following antibodies were from Santa Cruz Biotechnology: anti-NRP1 (C-19), anti-NRP2 (C-9), anti-Flk-1 (N-19), anti-synectin/GIPC [GAIP (Gα -interacting protein)-interacting protein C-terminus] (C-1158), PDGFRβ (PDGF receptor β), PDGFRα (C-20), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (V-18) and secondary antibodies against goat, rabbit and mouse.

    Techniques: Expressing, Flow Cytometry, Cytometry, Incubation, Western Blot, Modification

    Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line. DRMs were prepared from confluent PTr cells as described in Materials and Methods . Collected fractions, were concentrated, treated with heparitinase I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).

    Journal: PLoS ONE

    Article Title: Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

    doi: 10.1371/journal.pone.0032351

    Figure Lengend Snippet: Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line. DRMs were prepared from confluent PTr cells as described in Materials and Methods . Collected fractions, were concentrated, treated with heparitinase I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).

    Article Snippet: Biotinylated mouse anti-ΔHS (3G10) antibodies, recognizing HS neo-epitope, generated by the digestion with heparitinase I from Flavobacterium heparinum and heparitinase I (Flavobacterium heparinum ) were purchased from Seikagaku Corporation, (Tokyo, Japan).

    Techniques: Western Blot, Isolation, SDS Page, Staining, Concentration Assay

    Identification of HSPGs expressed by a rat parathyroid cell line. A. RT-PCR analysis of PTr cells using syndecan-specific primers (see Materials and Methods for details). Total RNA was isolated from confluent cells and subjected to RT-PCR analysis. Amplified products were run on 2% agarose gel, stained with ethidium bromide and photographed under UV transilluminator. Lanes: M – 100 bp marker; SN1 – amplification with syndecan-1 specific primers; SN2 – amplification with syndecan-2 specific primers; SN3 – amplification with syndecan-3 specific primers; SN4 – amplification with syndecan-4 specific primers; G – amplification with GAPHD specific primers; (-) – negative controls containing no cDNA. B. Identification of HSPGs present in DRM fractions using WB analysis. Proteoglycans were isolated from confluent rat parathyroid cells and partially purified using Q-Sepharose anion-exchange chromatography. A proteoglycan-enriched fraction was incubated in the presence or absence of heparitinase I, subjected to SDS-PAGE and immunoblotted with anti-syndecan-1, anti-syndecan-4 or anti-ΔHS (3G10) antibodies. Lanes: 1, 4 and 7 represent the heparitinase I only; 2, 5 and 8 correspond to the control samples, incubated without heparitinase I; 3, 6, 8 correspond to the heparitinase-treated samples.

    Journal: PLoS ONE

    Article Title: Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

    doi: 10.1371/journal.pone.0032351

    Figure Lengend Snippet: Identification of HSPGs expressed by a rat parathyroid cell line. A. RT-PCR analysis of PTr cells using syndecan-specific primers (see Materials and Methods for details). Total RNA was isolated from confluent cells and subjected to RT-PCR analysis. Amplified products were run on 2% agarose gel, stained with ethidium bromide and photographed under UV transilluminator. Lanes: M – 100 bp marker; SN1 – amplification with syndecan-1 specific primers; SN2 – amplification with syndecan-2 specific primers; SN3 – amplification with syndecan-3 specific primers; SN4 – amplification with syndecan-4 specific primers; G – amplification with GAPHD specific primers; (-) – negative controls containing no cDNA. B. Identification of HSPGs present in DRM fractions using WB analysis. Proteoglycans were isolated from confluent rat parathyroid cells and partially purified using Q-Sepharose anion-exchange chromatography. A proteoglycan-enriched fraction was incubated in the presence or absence of heparitinase I, subjected to SDS-PAGE and immunoblotted with anti-syndecan-1, anti-syndecan-4 or anti-ΔHS (3G10) antibodies. Lanes: 1, 4 and 7 represent the heparitinase I only; 2, 5 and 8 correspond to the control samples, incubated without heparitinase I; 3, 6, 8 correspond to the heparitinase-treated samples.

    Article Snippet: Biotinylated mouse anti-ΔHS (3G10) antibodies, recognizing HS neo-epitope, generated by the digestion with heparitinase I from Flavobacterium heparinum and heparitinase I (Flavobacterium heparinum ) were purchased from Seikagaku Corporation, (Tokyo, Japan).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Purification, Chromatography, Incubation, SDS Page

    Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.

    Journal: The Journal of Cell Biology

    Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

    doi:

    Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.

    Article Snippet: Although trypsin treatment completely inhibited the reaction (Fig. , bottom right ), activated BAEC exposure to heparinase I, heparitinase II, or heparitinase III only had moderate inhibitory effects on L-selectin binding (Fig. , top right and middle ).

    Techniques: Immunofluorescence

    Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated BAEC with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/μ were as follows: control, 86%; heparinase I, 54%; heparitinase II, 56%; chondroitinase, 89%; hyaluronidase, 90%; trypsin, 7%. The background staining with CD4/μ chimera was

    Journal: The Journal of Cell Biology

    Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

    doi:

    Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated BAEC with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/μ were as follows: control, 86%; heparinase I, 54%; heparitinase II, 56%; chondroitinase, 89%; hyaluronidase, 90%; trypsin, 7%. The background staining with CD4/μ chimera was

    Article Snippet: Although trypsin treatment completely inhibited the reaction (Fig. , bottom right ), activated BAEC exposure to heparinase I, heparitinase II, or heparitinase III only had moderate inhibitory effects on L-selectin binding (Fig. , top right and middle ).

    Techniques: Immunofluorescence, Staining

    Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase ABC/heparitinase digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.

    Journal: Developmental Dynamics

    Article Title: Expression of Glycosaminoglycan Epitopes During Zebrafish Skeletogenesis

    doi: 10.1002/dvdy.23970

    Figure Lengend Snippet: Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase ABC/heparitinase digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.

    Article Snippet: To generate the reactive HS neoepitope recognised by mAb 3G10, larvae were pre-digested with 5 mU/ml heparitinase (Amsbio, Lake Forest, CA) in 50 mM sodium acetate buffer (pH 7.0) containing 5 mM CaCl2 for 1 hr at 37°C, and re-fixed in 4% PFA.

    Techniques: Staining, Immunohistochemistry, Fluorescence In Situ Hybridization, Binding Assay

    Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

    Journal: The American Journal of Pathology

    Article Title: Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

    doi: 10.2353/ajpath.2009.081129

    Figure Lengend Snippet: Co-immunoprecipitation of CD81 and GPC3. The rat liver lysates without heparitinase treatment were incubated with anti-CD81 monoclonal antibody or control IgG, followed by precipitation with agarose A/G plus beads. Precipitates were separated by Western

    Article Snippet: For HS chain elimination, 100 μg of protein extract from freshly isolated rat hepatocytes and total rat liver was treated with 5 microunits of heparitinase enzyme (Seikagaku, Tokyo, Japan) and 1 mmol/L CaCl2 for 3 hours.

    Techniques: Immunoprecipitation, Incubation, Western Blot

    The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of heparitinase treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.

    Journal: The American Journal of Pathology

    Article Title: Expression of c-Met and Heparan-Sulfate Proteoglycan Forms of CD44 in Colorectal Cancer

    doi:

    Figure Lengend Snippet: The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of heparitinase treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.

    Article Snippet: For enzymatic cleavage of GAGs, cells were treated with either heparitinase ( Flafobacterium heparinum , EC 4.2.2.8; ICN Biomedicals, Aurora, OH) or chondroitinase avidin-biotin-peroxidase complex ( Proteus vulgaris , EC 4.2.2.4; Boehringer Mannheim, Almere, The Netherlands) in phosphate-buffered saline (PBS) at 37°C for the periods indicated.

    Techniques: Immunoprecipitation, Activation Assay, Binding Assay, Mutagenesis

    CD44v3 isoforms on colon carcinoma cell lines are decorated with HS. CD44v3 was immunoprecipitated from the colon carcinoma cell lines SW480 and HT-29, and, as a positive control, from Namalwa cells transfected with CD44v3–10, with mouse anti-CD44v3. Before immunoprecipitation, the cells were treated with either PBS (−), 30 mU/ml heparitinase (HT), or 30 mU/ml chondroitinase ABC (CH) at 37°C for 3.5 hours. The Western blot of the precipitates was stained with the anti-pan CD44 mAb Hermes-3, stripped, and restained with the mAb 3G10 that detects ΔHS stubs after treatment of HS with heparitinase. CD44v3 isoforms decorated with HS are indicated with arrows .

    Journal: The American Journal of Pathology

    Article Title: Expression of c-Met and Heparan-Sulfate Proteoglycan Forms of CD44 in Colorectal Cancer

    doi:

    Figure Lengend Snippet: CD44v3 isoforms on colon carcinoma cell lines are decorated with HS. CD44v3 was immunoprecipitated from the colon carcinoma cell lines SW480 and HT-29, and, as a positive control, from Namalwa cells transfected with CD44v3–10, with mouse anti-CD44v3. Before immunoprecipitation, the cells were treated with either PBS (−), 30 mU/ml heparitinase (HT), or 30 mU/ml chondroitinase ABC (CH) at 37°C for 3.5 hours. The Western blot of the precipitates was stained with the anti-pan CD44 mAb Hermes-3, stripped, and restained with the mAb 3G10 that detects ΔHS stubs after treatment of HS with heparitinase. CD44v3 isoforms decorated with HS are indicated with arrows .

    Article Snippet: For enzymatic cleavage of GAGs, cells were treated with either heparitinase ( Flafobacterium heparinum , EC 4.2.2.8; ICN Biomedicals, Aurora, OH) or chondroitinase avidin-biotin-peroxidase complex ( Proteus vulgaris , EC 4.2.2.4; Boehringer Mannheim, Almere, The Netherlands) in phosphate-buffered saline (PBS) at 37°C for the periods indicated.

    Techniques: Immunoprecipitation, Positive Control, Transfection, Western Blot, Staining

    HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.

    Journal: BMC Developmental Biology

    Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

    doi: 10.1186/1471-213X-11-38

    Figure Lengend Snippet: HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.

    Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 4.2.2.8) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.

    Techniques: Negative Control, Incubation

    Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.

    Journal: BMC Developmental Biology

    Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

    doi: 10.1186/1471-213X-11-38

    Figure Lengend Snippet: Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.

    Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 4.2.2.8) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.

    Techniques: Staining, Immunohistochemistry

    HS 'phage display antibodies identify distinct epitopes in situ . In fetal rat lungs, HS antibodies display different patterns of staining. HS3B7V exclusively labels epithelial basement membranes, whereas HS4E4V and HS3A8V show a more widespread staining pattern. In addition to epithelial basement membrane staining, HS4E4V labels sub-epithelial mesenchymal cells surrounding smaller distal airways and HS3A8V highlights the entire lung mesenchyme and in addition, stains epithelial cells at E15.5. One antibody, HS4C3V, did not stain fetal rat lungs of any developmental age; however, positive staining of adult rat kidney confirmed the functionality of HS4C3V in immunohistochemistry. E15.5 and E17.5 rat lungs and adult rat kidney were probed with HS antibodies followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Negative controls were omission of HS antibody or digestion of HS with heparitinase prior to antibody incubation (HS4E4V shown, heparitinase digest controls for other antibodies are shown in additional files). Scale bar represents 10 μm and all images are the same magnification. (ep) epithelium, (me) mesenchyme, (bm) basement membrane, (aw) airway, (G) glomerulus, (cap) peritubular capillary.

    Journal: BMC Developmental Biology

    Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

    doi: 10.1186/1471-213X-11-38

    Figure Lengend Snippet: HS 'phage display antibodies identify distinct epitopes in situ . In fetal rat lungs, HS antibodies display different patterns of staining. HS3B7V exclusively labels epithelial basement membranes, whereas HS4E4V and HS3A8V show a more widespread staining pattern. In addition to epithelial basement membrane staining, HS4E4V labels sub-epithelial mesenchymal cells surrounding smaller distal airways and HS3A8V highlights the entire lung mesenchyme and in addition, stains epithelial cells at E15.5. One antibody, HS4C3V, did not stain fetal rat lungs of any developmental age; however, positive staining of adult rat kidney confirmed the functionality of HS4C3V in immunohistochemistry. E15.5 and E17.5 rat lungs and adult rat kidney were probed with HS antibodies followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Negative controls were omission of HS antibody or digestion of HS with heparitinase prior to antibody incubation (HS4E4V shown, heparitinase digest controls for other antibodies are shown in additional files). Scale bar represents 10 μm and all images are the same magnification. (ep) epithelium, (me) mesenchyme, (bm) basement membrane, (aw) airway, (G) glomerulus, (cap) peritubular capillary.

    Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 4.2.2.8) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.

    Techniques: In Situ, Staining, Immunohistochemistry, Incubation

    Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Cell Culture, Activation Assay, Recombinant

    BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Binding Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Stable Transfection, Labeling, Plasmid Preparation

    Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page, Molecular Weight

    A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Binding Assay, Variant Assay, Activation Assay, Labeling

    A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Binding Assay, Variant Assay, Labeling, Activation Assay, Sample Prep, Positive Control

    TG2 extracellular location depends on the HS chains of HSPGs. Kidney cryosections were treated with 50 mU/ml protease-free heparitinase I (Hep-I) for 2 hours at 37°C. Extracellular TG2 was immunolabeled using mouse anti-TG2 IA12 antibody followed

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Syndecan-4 Knockout Leads to Reduced Extracellular Transglutaminase-2 and Protects against Tubulointerstitial Fibrosis

    doi: 10.1681/ASN.2013050563

    Figure Lengend Snippet: TG2 extracellular location depends on the HS chains of HSPGs. Kidney cryosections were treated with 50 mU/ml protease-free heparitinase I (Hep-I) for 2 hours at 37°C. Extracellular TG2 was immunolabeled using mouse anti-TG2 IA12 antibody followed

    Article Snippet: Proteinase-free heparitinase I (EC 4.2.2.8) (Sigma-Aldrich) was used to digest the side chains of HSPG.

    Techniques: Immunolabeling