heparitinases Search Results


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  • 99
    Millipore heparitinase iii
    Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with <t>heparitinase</t> <t>III,</t> as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P
    Heparitinase Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Seikagaku heparitinase mixture
    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 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
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    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 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    91
    ICN Biomedicals heparitinase
    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, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 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 Millipore, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Seikagaku flavobacterium heparinum heparitinase
    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.
    Flavobacterium Heparinum Heparitinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Seikagaku bacterial heparitinase
    Heparanase is expressed by HEK293T and HSG cells and is required for lacritin-dependent mitogenesis. (A) Lysates of HSG cells (lane 1) and HEK293T cells stably expressing human SDC1 (lanes 2) versus 2 M NaCl eluant of each after incubation with HiTrap heparin affinity columns (lanes 3 and 4, respectively). Blotting is with polyclonal anti–human heparanase-1 (HPSE1) antibody. (B) Lysates from HSG cells that had been mock transfected or transfected with 1 nM heparanase-1 siRNA. Blotting is with polyclonal anti–human HPSE1 or anti-tubulin antibodies. (C) Proliferation assay in which HSG cells were treated with 10 nM lacritin or 1 nM EGF 48 h after being mock transfected or transfected with 10 nM of Ambion's negative control siRNA #1 (neg), 1–100 nM HPSE1 siRNA, or 1 nM HPSE2 siRNA. Some HPSE1 siRNA cells were lacritin treated for 24 h in the presence of 1 μg of heparanase-enriched eluant (A) from HEK293T cells stably expressing SDC1 (1 nM + HPSE) or 0.0001 U of bacterial <t>heparitinase.</t> Error bars indicate SEM. (D) Sepharose CL-6B gel filtration chromatography of HS from lacritin and FGF2 affinity enriched SDC1 isolated from normal or HPSE1-depleted HSG cells. Lysates from cells labeled with 50 μCi/ml Na 2 35 SO 4 in DME for 48 h were affinity precipitated with FGF2-GST or lacritin-intein. Equal microgram amounts of SDC1 bound to beads were digested with chondroitin ABC lyase to remove CS, eluted with 2 M NaCl, and subjected to NaBH 4 eliminative cleavage. Released HS was neutralized by drop-wise addition of 1 M HCl and subjected to Sepharose CL-6B gel filtration chromatography to compare the relative size of HS chains. V 0 , void volume (dextran blue); V t , total volume (sodium dichromate).
    Bacterial Heparitinase, 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
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    85
    AMS Biotechnology heparitinase 1
    Heparanase is expressed by HEK293T and HSG cells and is required for lacritin-dependent mitogenesis. (A) Lysates of HSG cells (lane 1) and HEK293T cells stably expressing human SDC1 (lanes 2) versus 2 M NaCl eluant of each after incubation with HiTrap heparin affinity columns (lanes 3 and 4, respectively). Blotting is with polyclonal anti–human heparanase-1 (HPSE1) antibody. (B) Lysates from HSG cells that had been mock transfected or transfected with 1 nM heparanase-1 siRNA. Blotting is with polyclonal anti–human HPSE1 or anti-tubulin antibodies. (C) Proliferation assay in which HSG cells were treated with 10 nM lacritin or 1 nM EGF 48 h after being mock transfected or transfected with 10 nM of Ambion's negative control siRNA #1 (neg), 1–100 nM HPSE1 siRNA, or 1 nM HPSE2 siRNA. Some HPSE1 siRNA cells were lacritin treated for 24 h in the presence of 1 μg of heparanase-enriched eluant (A) from HEK293T cells stably expressing SDC1 (1 nM + HPSE) or 0.0001 U of bacterial <t>heparitinase.</t> Error bars indicate SEM. (D) Sepharose CL-6B gel filtration chromatography of HS from lacritin and FGF2 affinity enriched SDC1 isolated from normal or HPSE1-depleted HSG cells. Lysates from cells labeled with 50 μCi/ml Na 2 35 SO 4 in DME for 48 h were affinity precipitated with FGF2-GST or lacritin-intein. Equal microgram amounts of SDC1 bound to beads were digested with chondroitin ABC lyase to remove CS, eluted with 2 M NaCl, and subjected to NaBH 4 eliminative cleavage. Released HS was neutralized by drop-wise addition of 1 M HCl and subjected to Sepharose CL-6B gel filtration chromatography to compare the relative size of HS chains. V 0 , void volume (dextran blue); V t , total volume (sodium dichromate).
    Heparitinase 1, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Seikagaku heparitinase 3g10
    Heparanase is expressed by HEK293T and HSG cells and is required for lacritin-dependent mitogenesis. (A) Lysates of HSG cells (lane 1) and HEK293T cells stably expressing human SDC1 (lanes 2) versus 2 M NaCl eluant of each after incubation with HiTrap heparin affinity columns (lanes 3 and 4, respectively). Blotting is with polyclonal anti–human heparanase-1 (HPSE1) antibody. (B) Lysates from HSG cells that had been mock transfected or transfected with 1 nM heparanase-1 siRNA. Blotting is with polyclonal anti–human HPSE1 or anti-tubulin antibodies. (C) Proliferation assay in which HSG cells were treated with 10 nM lacritin or 1 nM EGF 48 h after being mock transfected or transfected with 10 nM of Ambion's negative control siRNA #1 (neg), 1–100 nM HPSE1 siRNA, or 1 nM HPSE2 siRNA. Some HPSE1 siRNA cells were lacritin treated for 24 h in the presence of 1 μg of heparanase-enriched eluant (A) from HEK293T cells stably expressing SDC1 (1 nM + HPSE) or 0.0001 U of bacterial <t>heparitinase.</t> Error bars indicate SEM. (D) Sepharose CL-6B gel filtration chromatography of HS from lacritin and FGF2 affinity enriched SDC1 isolated from normal or HPSE1-depleted HSG cells. Lysates from cells labeled with 50 μCi/ml Na 2 35 SO 4 in DME for 48 h were affinity precipitated with FGF2-GST or lacritin-intein. Equal microgram amounts of SDC1 bound to beads were digested with chondroitin ABC lyase to remove CS, eluted with 2 M NaCl, and subjected to NaBH 4 eliminative cleavage. Released HS was neutralized by drop-wise addition of 1 M HCl and subjected to Sepharose CL-6B gel filtration chromatography to compare the relative size of HS chains. V 0 , void volume (dextran blue); V t , total volume (sodium dichromate).
    Heparitinase 3g10, supplied by Seikagaku, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells

    doi: 10.1167/iovs.18-25202

    Figure Lengend Snippet: Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Article Snippet: For perlecan Western blots, the dialyzed extract was digested with 1 mU/mL of heparitinase III (Cat No. H8891; Sigma-Aldrich) at 37°C for 3 hours.

    Techniques: Expressing, Cell Culture, Western Blot

    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

    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

    Heparinase III treatment efficiently removes heparan sulfate from human tracheal epithelial (HTE) cell membranes . Cells were incubated with a monoclonal mouse anti-heparan sulfate (10E4 epitope) antibody followed by a Cy3 conjugated donkey anti-mouse IgG. A DAPI nuclear counterstain was applied. Treatment of HTE cells with 10 mIU and 20 mIU of heparinase III reduced membrane HSPG staining to levels seen in cells not treated with primary anti-heparan antibody.

    Journal: Virology Journal

    Article Title: Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia

    doi: 10.1186/1743-422X-3-29

    Figure Lengend Snippet: Heparinase III treatment efficiently removes heparan sulfate from human tracheal epithelial (HTE) cell membranes . Cells were incubated with a monoclonal mouse anti-heparan sulfate (10E4 epitope) antibody followed by a Cy3 conjugated donkey anti-mouse IgG. A DAPI nuclear counterstain was applied. Treatment of HTE cells with 10 mIU and 20 mIU of heparinase III reduced membrane HSPG staining to levels seen in cells not treated with primary anti-heparan antibody.

    Article Snippet: Reagents Glycosaminoglycan-specific enzymes heparinase III (heparitinase) (H8891) and chondroitinase ABC (C3667) were purchased from Sigma (St. Louis, MO).

    Techniques: Incubation, Staining

    Heparinase III treatment efficiently removes heparan sulfate from respiratory epithelial cell membranes . Immortalized epithelial cell lines fetal human tracheal (FHTE), human tracheal (HTE), and cystic fibrosis bronchial (IB3-1) were treated with increasing amounts of heparinase III for 60 minutes, incubated with Cy3-conjugated mouse anti-heparan sulfate antibody (10E4 epitope), and assessed for HSPG surface expression by flow cytometry. Each experiment was done in triplicate and expression was normalized to mean untreated expression level. Removal of surface HSPG plateaued after treatment with 10 mIU.

    Journal: Virology Journal

    Article Title: Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia

    doi: 10.1186/1743-422X-3-29

    Figure Lengend Snippet: Heparinase III treatment efficiently removes heparan sulfate from respiratory epithelial cell membranes . Immortalized epithelial cell lines fetal human tracheal (FHTE), human tracheal (HTE), and cystic fibrosis bronchial (IB3-1) were treated with increasing amounts of heparinase III for 60 minutes, incubated with Cy3-conjugated mouse anti-heparan sulfate antibody (10E4 epitope), and assessed for HSPG surface expression by flow cytometry. Each experiment was done in triplicate and expression was normalized to mean untreated expression level. Removal of surface HSPG plateaued after treatment with 10 mIU.

    Article Snippet: Reagents Glycosaminoglycan-specific enzymes heparinase III (heparitinase) (H8891) and chondroitinase ABC (C3667) were purchased from Sigma (St. Louis, MO).

    Techniques: Incubation, Expressing, Flow Cytometry, Cytometry

    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

    Adhesion of the B16V melanoma cell lines to fibronectin. ( A ) Adhesion of B16V control, B16V shUst(6) and B16V shUst(16) cells in fibronectin-coated wells. ( B ) Adhesion of B16V control and B16V shUst(16) cells to fibronectin after 1h after treatment with chondroitin ABC layse (ABCase), heparitinase (Hep-Mix) and ABCase+Hep-Mix. ( C ) Adhesion of B16V and B16V shUst(16) cells to fibronectin for 1h after blocking of αvβ3 integrin. The integrins were blocked with the αv integrin blocking antibody, isotype control IC-1 (Rat IgG1, κ as control against αv integrin), β3 integrin blocking antibody and isotype control IC-2 (Armenian Hamster IgG towards β3 integrin). ( D ) Adhesion of B16V and B16V shUst(16) cells to fibronectin after blocking with α5 integrin blocking antibody, isotype control IC-3 (Rat IgG2a, κ as control for α5 integrin) and β1 integrin blocking antibody and isotype control IC-2 (Armenian Hamster IgG for β1 integrin). Each experiment was performed in duplicates, n = 3, mean±SD ( A , B ), mean±SEM ( C , D ), *, P

    Journal: PLoS ONE

    Article Title: Melanoma Cell Adhesion and Migration Is Modulated by the Uronyl 2-O Sulfotransferase

    doi: 10.1371/journal.pone.0170054

    Figure Lengend Snippet: Adhesion of the B16V melanoma cell lines to fibronectin. ( A ) Adhesion of B16V control, B16V shUst(6) and B16V shUst(16) cells in fibronectin-coated wells. ( B ) Adhesion of B16V control and B16V shUst(16) cells to fibronectin after 1h after treatment with chondroitin ABC layse (ABCase), heparitinase (Hep-Mix) and ABCase+Hep-Mix. ( C ) Adhesion of B16V and B16V shUst(16) cells to fibronectin for 1h after blocking of αvβ3 integrin. The integrins were blocked with the αv integrin blocking antibody, isotype control IC-1 (Rat IgG1, κ as control against αv integrin), β3 integrin blocking antibody and isotype control IC-2 (Armenian Hamster IgG towards β3 integrin). ( D ) Adhesion of B16V and B16V shUst(16) cells to fibronectin after blocking with α5 integrin blocking antibody, isotype control IC-3 (Rat IgG2a, κ as control for α5 integrin) and β1 integrin blocking antibody and isotype control IC-2 (Armenian Hamster IgG for β1 integrin). Each experiment was performed in duplicates, n = 3, mean±SD ( A , B ), mean±SEM ( C , D ), *, P

    Article Snippet: PD173074, fibronectin, mouse-Fgf2, chondroitin 6-sulfate (CS-6S) (Sigma Aldrich, Deisenhofen, Germany), chondroitin ABC lyase and heparitinase mix (heparinase II/III, 4:1) (Amsbio, UK).

    Techniques: Blocking Assay

    Organization and Differentiation of Endodermal Cells is Dependent on HS Proteoglycans on Decellularized Lung Scaffolds (A) Acellular scaffolds were recellularized after heparitinase I or chondroitinase ABC treatment. H E staining of day 21 seeded scaffold cultures show limited organization and differentiation in the heparitinase I-treated group, while chondroitinase ABC-treated cultures resemble control groups. Scale bar represents 50 μm. (B) Scanning EM analysis of cultures show a lack of epithelial morphology and tight junction coupling of seeded cells in heparitinase I-treated scaffolds, where cells appear rounded with no resemblance to a lung phenotype. Scale bar represents 10 μm. (C) Proteome profiler antibody array detects 31 proteins from the array profile that are remaining on lung scaffolds (black). Comparison of the protein profile from decellularized scaffolds treated with or without heparitinase I revealed several HS-bound proteins that are removed from scaffolds and found in the wash supernatant after enzyme treatment (red rectangles): CXCL12, serpinE1, PDGF-AB, HGF, MMP8, FGF2, proliferin, IL10, and CCL3. Data presented are average of two arrays from separate experiments. See also Figure S5 .

    Journal: Stem Cell Reports

    Article Title: Acellular Lung Scaffolds Direct Differentiation of Endoderm to Functional Airway Epithelial Cells: Requirement of Matrix-Bound HS Proteoglycans

    doi: 10.1016/j.stemcr.2015.01.004

    Figure Lengend Snippet: Organization and Differentiation of Endodermal Cells is Dependent on HS Proteoglycans on Decellularized Lung Scaffolds (A) Acellular scaffolds were recellularized after heparitinase I or chondroitinase ABC treatment. H E staining of day 21 seeded scaffold cultures show limited organization and differentiation in the heparitinase I-treated group, while chondroitinase ABC-treated cultures resemble control groups. Scale bar represents 50 μm. (B) Scanning EM analysis of cultures show a lack of epithelial morphology and tight junction coupling of seeded cells in heparitinase I-treated scaffolds, where cells appear rounded with no resemblance to a lung phenotype. Scale bar represents 10 μm. (C) Proteome profiler antibody array detects 31 proteins from the array profile that are remaining on lung scaffolds (black). Comparison of the protein profile from decellularized scaffolds treated with or without heparitinase I revealed several HS-bound proteins that are removed from scaffolds and found in the wash supernatant after enzyme treatment (red rectangles): CXCL12, serpinE1, PDGF-AB, HGF, MMP8, FGF2, proliferin, IL10, and CCL3. Data presented are average of two arrays from separate experiments. See also Figure S5 .

    Article Snippet: Scaffold Enzymatic Treatment HS or CS proteoglycans were cleaved from decellularized lung scaffolds using treatment with heparitinase I (Amsbio #100704) or chondroitinase ABC (Amsbio #100330-1A), respectively.

    Techniques: Staining, Ab Array

    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

    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

    Heparanase is expressed by HEK293T and HSG cells and is required for lacritin-dependent mitogenesis. (A) Lysates of HSG cells (lane 1) and HEK293T cells stably expressing human SDC1 (lanes 2) versus 2 M NaCl eluant of each after incubation with HiTrap heparin affinity columns (lanes 3 and 4, respectively). Blotting is with polyclonal anti–human heparanase-1 (HPSE1) antibody. (B) Lysates from HSG cells that had been mock transfected or transfected with 1 nM heparanase-1 siRNA. Blotting is with polyclonal anti–human HPSE1 or anti-tubulin antibodies. (C) Proliferation assay in which HSG cells were treated with 10 nM lacritin or 1 nM EGF 48 h after being mock transfected or transfected with 10 nM of Ambion's negative control siRNA #1 (neg), 1–100 nM HPSE1 siRNA, or 1 nM HPSE2 siRNA. Some HPSE1 siRNA cells were lacritin treated for 24 h in the presence of 1 μg of heparanase-enriched eluant (A) from HEK293T cells stably expressing SDC1 (1 nM + HPSE) or 0.0001 U of bacterial heparitinase. Error bars indicate SEM. (D) Sepharose CL-6B gel filtration chromatography of HS from lacritin and FGF2 affinity enriched SDC1 isolated from normal or HPSE1-depleted HSG cells. Lysates from cells labeled with 50 μCi/ml Na 2 35 SO 4 in DME for 48 h were affinity precipitated with FGF2-GST or lacritin-intein. Equal microgram amounts of SDC1 bound to beads were digested with chondroitin ABC lyase to remove CS, eluted with 2 M NaCl, and subjected to NaBH 4 eliminative cleavage. Released HS was neutralized by drop-wise addition of 1 M HCl and subjected to Sepharose CL-6B gel filtration chromatography to compare the relative size of HS chains. V 0 , void volume (dextran blue); V t , total volume (sodium dichromate).

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Heparanase is expressed by HEK293T and HSG cells and is required for lacritin-dependent mitogenesis. (A) Lysates of HSG cells (lane 1) and HEK293T cells stably expressing human SDC1 (lanes 2) versus 2 M NaCl eluant of each after incubation with HiTrap heparin affinity columns (lanes 3 and 4, respectively). Blotting is with polyclonal anti–human heparanase-1 (HPSE1) antibody. (B) Lysates from HSG cells that had been mock transfected or transfected with 1 nM heparanase-1 siRNA. Blotting is with polyclonal anti–human HPSE1 or anti-tubulin antibodies. (C) Proliferation assay in which HSG cells were treated with 10 nM lacritin or 1 nM EGF 48 h after being mock transfected or transfected with 10 nM of Ambion's negative control siRNA #1 (neg), 1–100 nM HPSE1 siRNA, or 1 nM HPSE2 siRNA. Some HPSE1 siRNA cells were lacritin treated for 24 h in the presence of 1 μg of heparanase-enriched eluant (A) from HEK293T cells stably expressing SDC1 (1 nM + HPSE) or 0.0001 U of bacterial heparitinase. Error bars indicate SEM. (D) Sepharose CL-6B gel filtration chromatography of HS from lacritin and FGF2 affinity enriched SDC1 isolated from normal or HPSE1-depleted HSG cells. Lysates from cells labeled with 50 μCi/ml Na 2 35 SO 4 in DME for 48 h were affinity precipitated with FGF2-GST or lacritin-intein. Equal microgram amounts of SDC1 bound to beads were digested with chondroitin ABC lyase to remove CS, eluted with 2 M NaCl, and subjected to NaBH 4 eliminative cleavage. Released HS was neutralized by drop-wise addition of 1 M HCl and subjected to Sepharose CL-6B gel filtration chromatography to compare the relative size of HS chains. V 0 , void volume (dextran blue); V t , total volume (sodium dichromate).

    Article Snippet: To rescue heparanase-depleted cells, ∼1 μg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 U bacterial heparitinase (Seikagaku America) was added together with lacritin and [3 H]-thymidine for 24 h. [3 H]-thymidine incorporation was stopped by placing on ice.

    Techniques: Stable Transfection, Expressing, Incubation, Transfection, Proliferation Assay, Negative Control, Filtration, Chromatography, Isolation, Labeling

    Lacritin binding to SDC1 is independent of complete HS/CS glycosaminoglycans. (A) Lacritin affinity precipitation of human SDC1 multimers stably expressed by HEK293T cells. Lacritin-intein beads were incubated with cell lysates, washed extensively, and treated with heparitinase I/chondroitinase ABC. Pellet (P) and supernatant (S) from the centrifuged digest were then blotted with mAb B-B4 for SDC1 core protein. (B) Lacritin-intein, lacritin-GST, FGF2-GST, intein, and GST beads were incubated with lysates from the same HEK293T cells stably expressing human SDC1. Precipitates were washed, treated, centrifuged, and blotted as in A. (C) Lacritin-intein and FGF2-GST beads were incubated with lysate of HEK293T cells stably expressing human SDC2 or lysate of another HEK293T cell line stably expressing human SDC4. Beads were washed, treated, and centrifuged as in A. Blots were detected with anti-SDC2 mAb L-18 or anti-SDC4 mAb N-19, respectively. A shows both 190- and 80-kD bands. B and C and all subsequent figures show the 80-kD band, which is more predominant in HEK293T transfectants.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin binding to SDC1 is independent of complete HS/CS glycosaminoglycans. (A) Lacritin affinity precipitation of human SDC1 multimers stably expressed by HEK293T cells. Lacritin-intein beads were incubated with cell lysates, washed extensively, and treated with heparitinase I/chondroitinase ABC. Pellet (P) and supernatant (S) from the centrifuged digest were then blotted with mAb B-B4 for SDC1 core protein. (B) Lacritin-intein, lacritin-GST, FGF2-GST, intein, and GST beads were incubated with lysates from the same HEK293T cells stably expressing human SDC1. Precipitates were washed, treated, centrifuged, and blotted as in A. (C) Lacritin-intein and FGF2-GST beads were incubated with lysate of HEK293T cells stably expressing human SDC2 or lysate of another HEK293T cell line stably expressing human SDC4. Beads were washed, treated, and centrifuged as in A. Blots were detected with anti-SDC2 mAb L-18 or anti-SDC4 mAb N-19, respectively. A shows both 190- and 80-kD bands. B and C and all subsequent figures show the 80-kD band, which is more predominant in HEK293T transfectants.

    Article Snippet: To rescue heparanase-depleted cells, ∼1 μg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 U bacterial heparitinase (Seikagaku America) was added together with lacritin and [3 H]-thymidine for 24 h. [3 H]-thymidine incorporation was stopped by placing on ice.

    Techniques: Binding Assay, Affinity Precipitation, Stable Transfection, Incubation, Expressing

    Bacterial heparitinase digestion exposes FGF2-bindable SDC1 to lacritin binding via a domain in SDC1's N-terminal 50 amino acids. (A) Human SDC1 (lanes 1 and 2), SDC2 (lanes 3 and 4), and SDC4 (lanes 5 and 6) from stably expressing HEK293T cells were individually purified on FGF2-GST, eluted (0.5 and 1 M NaCl), treated with heparitinase I/chondroitinase ABC for 2 h, and incubated with lacritin-intein beads. Blotting is with mAb B-B4 for SDC1, polyclonal antibody L-18 for SDC2, or polyclonal N-19 for SDC4—all core protein specific. (B) Schematic diagram of human SDC1. The dotted line indicates truncation sites in the ectodomain forming the deletion constructs del 1–51, 51–252, and 51–310. Boxes represent PSIPRED-predicted α helices. Wavy lines represent HS and CS. TM, transmembrane domain. (C) Comparative incubation of FGF2-GST and lacritin-intein beads with human SDC1 or human SDC1 del 1–51 lysates from stably expressing HEK293T cells. After incubation, beads were washed extensively and either treated with heparitinase I/chondroitinase ABC (+) or left untreated (−). Beads were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Lysate from HEK293T cells stably expressing SDC1 del 1–51 is blotted in lanes 6 and 7. (D) Comparative incubation of lacritin-intein beads with human SDC1 del 51–252, 1–51, or 51–310 lysates from stably or transiently expressing HEK293T cells. pcDNA is lysate from cells transfected with vector only. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. Beads were centrifuged, and pellets were blotted with mAb 3G10 for desaturated uronates in SDC1.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Bacterial heparitinase digestion exposes FGF2-bindable SDC1 to lacritin binding via a domain in SDC1's N-terminal 50 amino acids. (A) Human SDC1 (lanes 1 and 2), SDC2 (lanes 3 and 4), and SDC4 (lanes 5 and 6) from stably expressing HEK293T cells were individually purified on FGF2-GST, eluted (0.5 and 1 M NaCl), treated with heparitinase I/chondroitinase ABC for 2 h, and incubated with lacritin-intein beads. Blotting is with mAb B-B4 for SDC1, polyclonal antibody L-18 for SDC2, or polyclonal N-19 for SDC4—all core protein specific. (B) Schematic diagram of human SDC1. The dotted line indicates truncation sites in the ectodomain forming the deletion constructs del 1–51, 51–252, and 51–310. Boxes represent PSIPRED-predicted α helices. Wavy lines represent HS and CS. TM, transmembrane domain. (C) Comparative incubation of FGF2-GST and lacritin-intein beads with human SDC1 or human SDC1 del 1–51 lysates from stably expressing HEK293T cells. After incubation, beads were washed extensively and either treated with heparitinase I/chondroitinase ABC (+) or left untreated (−). Beads were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Lysate from HEK293T cells stably expressing SDC1 del 1–51 is blotted in lanes 6 and 7. (D) Comparative incubation of lacritin-intein beads with human SDC1 del 51–252, 1–51, or 51–310 lysates from stably or transiently expressing HEK293T cells. pcDNA is lysate from cells transfected with vector only. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. Beads were centrifuged, and pellets were blotted with mAb 3G10 for desaturated uronates in SDC1.

    Article Snippet: To rescue heparanase-depleted cells, ∼1 μg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 U bacterial heparitinase (Seikagaku America) was added together with lacritin and [3 H]-thymidine for 24 h. [3 H]-thymidine incorporation was stopped by placing on ice.

    Techniques: Binding Assay, Stable Transfection, Expressing, Purification, Incubation, Construct, Transfection, Plasmid Preparation

    Lacritin's C terminus binds SDC1. (A) Schematic diagram of lacritin with dotted lines indicating N- and C-terminal truncations. All lacritin truncations were expressed as intein fusion proteins for affinity precipitation. The asterisk indicates mitogenic domain ( Wang et al., 2006 ), and boxes represent PSIPRED-predicted α helices. (B) Lacritin-, C-5–, C-10–, C-15–, C-25–, and C-59–intein beads were incubated with lysates from HEK293T cells stably expressing human SDC1. Beads were washed and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) blotted with mAb B-B4 for SDC1 core protein, all as in Fig. 2 . (C) Incubation of lacritin-, N-15– and N-24–intein beads with the same human SDC1 lysates was followed with identical washing, heparitinase I/chondroitinase ABC digestion, centrifugation, and B-B4 mAb blotting. Lys, lysate.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin's C terminus binds SDC1. (A) Schematic diagram of lacritin with dotted lines indicating N- and C-terminal truncations. All lacritin truncations were expressed as intein fusion proteins for affinity precipitation. The asterisk indicates mitogenic domain ( Wang et al., 2006 ), and boxes represent PSIPRED-predicted α helices. (B) Lacritin-, C-5–, C-10–, C-15–, C-25–, and C-59–intein beads were incubated with lysates from HEK293T cells stably expressing human SDC1. Beads were washed and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) blotted with mAb B-B4 for SDC1 core protein, all as in Fig. 2 . (C) Incubation of lacritin-, N-15– and N-24–intein beads with the same human SDC1 lysates was followed with identical washing, heparitinase I/chondroitinase ABC digestion, centrifugation, and B-B4 mAb blotting. Lys, lysate.

    Article Snippet: To rescue heparanase-depleted cells, ∼1 μg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 U bacterial heparitinase (Seikagaku America) was added together with lacritin and [3 H]-thymidine for 24 h. [3 H]-thymidine incorporation was stopped by placing on ice.

    Techniques: Affinity Precipitation, Incubation, Stable Transfection, Expressing, Centrifugation

    Lacritin-SDC1 binding is inhibited by soluble hS1ED, lacritin, and N-24, but not by C-25, C-59, HS, CS, SDC2, or SDC4. (A, top) Lacritin-intein beads were incubated with human SDC1 lysates from stably expressing HEK293T cells in the presence of increasing amounts of soluble HS (70–700 μg), HS (700 μg) plus CS (700 μg), or lacritin (14–700 μg) or no inhibitor (−). The quantity of soluble inhibitor was calibrated relative to the ∼7–8 μg of human SDC1 elutable from lacritin-intein beads with 1 M NaCl. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets were blotted with mAb B-B4 for SDC1 core protein, as in Fig. 2 . (bottom) Lacritin-intein beads were incubated with human SDC1 lysates in the presence of soluble N-24, C-25, C-59 (14 μg of each), increasing amounts of bacterially expressed human SDC1 ectodomain (hS1ED; 35–700 μg), or with HEK293T cell–expressed native SDC2 or -4 (70 μg of each). Beads were washed and treated as above. (B) Quantification of inhibition binding. Error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin-SDC1 binding is inhibited by soluble hS1ED, lacritin, and N-24, but not by C-25, C-59, HS, CS, SDC2, or SDC4. (A, top) Lacritin-intein beads were incubated with human SDC1 lysates from stably expressing HEK293T cells in the presence of increasing amounts of soluble HS (70–700 μg), HS (700 μg) plus CS (700 μg), or lacritin (14–700 μg) or no inhibitor (−). The quantity of soluble inhibitor was calibrated relative to the ∼7–8 μg of human SDC1 elutable from lacritin-intein beads with 1 M NaCl. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets were blotted with mAb B-B4 for SDC1 core protein, as in Fig. 2 . (bottom) Lacritin-intein beads were incubated with human SDC1 lysates in the presence of soluble N-24, C-25, C-59 (14 μg of each), increasing amounts of bacterially expressed human SDC1 ectodomain (hS1ED; 35–700 μg), or with HEK293T cell–expressed native SDC2 or -4 (70 μg of each). Beads were washed and treated as above. (B) Quantification of inhibition binding. Error bars indicate SEM.

    Article Snippet: To rescue heparanase-depleted cells, ∼1 μg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 U bacterial heparitinase (Seikagaku America) was added together with lacritin and [3 H]-thymidine for 24 h. [3 H]-thymidine incorporation was stopped by placing on ice.

    Techniques: Binding Assay, Incubation, Stable Transfection, Expressing, Inhibition

    Lacritin and FGF2 bind different forms of cell surface SDC1. (A) Sequential affinity precipitation assays. Lanes 1–3 show lysate from human SDC1 stably expressing HEK293T cells sequentially incubated with three rounds of fresh FGF2-GST beads. Half of the final depleted lysate was then incubated with lacritin-intein beads (lane 4), and the other half was methanol precipitated (lane 9). Similarly, in lanes 5–7, a different aliquot of lysate from the same cells was sequentially incubated with three rounds of fresh lacritin-intein beads. Half of the final depleted lysate was then incubated with FGF2-GST beads (lane 8), and the other half was methanol precipitated (lane 10). Beads were washed and treated with heparitinase I/ chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Shown are digest supernatants (lanes 1–3 and 8) and pellets (lanes 4–7) as per heparitinase release of FGF2-bound or resistance of lacritin-bound SDC1, respectively. (B) HEK293T cells stably expressing human SDC1 were either lysed as usual or first briefly trypsinized (

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin and FGF2 bind different forms of cell surface SDC1. (A) Sequential affinity precipitation assays. Lanes 1–3 show lysate from human SDC1 stably expressing HEK293T cells sequentially incubated with three rounds of fresh FGF2-GST beads. Half of the final depleted lysate was then incubated with lacritin-intein beads (lane 4), and the other half was methanol precipitated (lane 9). Similarly, in lanes 5–7, a different aliquot of lysate from the same cells was sequentially incubated with three rounds of fresh lacritin-intein beads. Half of the final depleted lysate was then incubated with FGF2-GST beads (lane 8), and the other half was methanol precipitated (lane 10). Beads were washed and treated with heparitinase I/ chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Shown are digest supernatants (lanes 1–3 and 8) and pellets (lanes 4–7) as per heparitinase release of FGF2-bound or resistance of lacritin-bound SDC1, respectively. (B) HEK293T cells stably expressing human SDC1 were either lysed as usual or first briefly trypsinized (

    Article Snippet: To rescue heparanase-depleted cells, ∼1 μg heparanase enriched from HSG or HEK293 cells using heparin affinity column or 0.0001 U bacterial heparitinase (Seikagaku America) was added together with lacritin and [3 H]-thymidine for 24 h. [3 H]-thymidine incorporation was stopped by placing on ice.

    Techniques: Affinity Precipitation, Stable Transfection, Expressing, Incubation

    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