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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 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku heparitinase
    Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by <t>heparitinase</t> treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p
    Heparitinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    AMS Biotechnology heparitinase
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not <t>heparitinase-treated</t> syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
    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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    85
    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: 85/100, 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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    91
    Associates of Cape Cod Inc heparitinase
    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, supplied by Associates of Cape Cod Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Isolation, Purification, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry, Cytometry, Affinity Purification, Western Blot

    Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Purification, Magnetic Beads, Flow Cytometry, Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

    Model of exosome-target cell interaction mediated by fibronectin. Step 1 , fibronectin ( FN ) is captured on the surface of exosomes by heparan sulfate proteoglycans ( HSPG ). Step 2 , fibronectin, on exosomes binds to heparan sulfate ( HS ) on the surface of the target cell. Step 3 , removal of exosome heparan sulfate or cell surface heparan sulfate with heparitinase, addition of exogenous heparin or heparin mimetics, addition of Hep-II domain-containing fragment of fibronectin, or exposure to antibody against the Hep-II domain of fibronectin dramatically diminishes exosome interaction with target cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Model of exosome-target cell interaction mediated by fibronectin. Step 1 , fibronectin ( FN ) is captured on the surface of exosomes by heparan sulfate proteoglycans ( HSPG ). Step 2 , fibronectin, on exosomes binds to heparan sulfate ( HS ) on the surface of the target cell. Step 3 , removal of exosome heparan sulfate or cell surface heparan sulfate with heparitinase, addition of exogenous heparin or heparin mimetics, addition of Hep-II domain-containing fragment of fibronectin, or exposure to antibody against the Hep-II domain of fibronectin dramatically diminishes exosome interaction with target cells.

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques:

    Exosome-target cell interaction mediated by fibronectin impacts cell behavior. A , exosome interaction with RPMI-8226 cells activates p38 and ERK signaling. An antibody array that simultaneously examines the phosphorylation levels of 43 different protein kinases was utilized to determine what signaling pathways were activated when myeloma-derived exosomes interacted with target cells. RPMI-8226 cells were incubated with or without exosomes isolated from aggressive myeloma cells (CAG cells expressing high heparanase), cell lysates were exposed to membranes, and the membranes were probed with a phosphotyrosine specific antibody. Phosphorylated p38 and ERK ( circled ) were enhanced in cells incubated with the exosomes. The different phospho kinases that are activated in RPMI-8226 cells independent of the addition of exosomes are shown by arrows. Duplicate dots at the corners represent phosphotyrosine positive controls. B , RPMI-8226 cells were treated with exosomes (100 μg/ml) secreted by control or aggressive myeloma cells for 20 min and analyzed for phosphorylated p38 by Western blot. Total p38 serves as the loading control. To determine the role of cell surface heparan sulfate in mediating exosome-induced signaling, RPMI-8266 cells were treated with bacterial heparitinase for 2 h prior to the addition of exosomes from aggressive myeloma cells. C , MMP-9 and DKK1, two downstream target genes of activated p38, are up-regulated following the interaction of myeloma-derived exosomes with the RPMI 8226 cells. RPMI 8266 myeloma cells were incubated with or without exosomes, and DKK1 and MMP-9 mRNA expression in these cells was assessed using real time PCR and normalized using actin expression. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Exosome-target cell interaction mediated by fibronectin impacts cell behavior. A , exosome interaction with RPMI-8226 cells activates p38 and ERK signaling. An antibody array that simultaneously examines the phosphorylation levels of 43 different protein kinases was utilized to determine what signaling pathways were activated when myeloma-derived exosomes interacted with target cells. RPMI-8226 cells were incubated with or without exosomes isolated from aggressive myeloma cells (CAG cells expressing high heparanase), cell lysates were exposed to membranes, and the membranes were probed with a phosphotyrosine specific antibody. Phosphorylated p38 and ERK ( circled ) were enhanced in cells incubated with the exosomes. The different phospho kinases that are activated in RPMI-8226 cells independent of the addition of exosomes are shown by arrows. Duplicate dots at the corners represent phosphotyrosine positive controls. B , RPMI-8226 cells were treated with exosomes (100 μg/ml) secreted by control or aggressive myeloma cells for 20 min and analyzed for phosphorylated p38 by Western blot. Total p38 serves as the loading control. To determine the role of cell surface heparan sulfate in mediating exosome-induced signaling, RPMI-8266 cells were treated with bacterial heparitinase for 2 h prior to the addition of exosomes from aggressive myeloma cells. C , MMP-9 and DKK1, two downstream target genes of activated p38, are up-regulated following the interaction of myeloma-derived exosomes with the RPMI 8226 cells. RPMI 8266 myeloma cells were incubated with or without exosomes, and DKK1 and MMP-9 mRNA expression in these cells was assessed using real time PCR and normalized using actin expression. *, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Ab Array, Derivative Assay, Incubation, Isolation, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Exosome-target cell interaction is mediated by heparan sulfate chains on target cells. A , confocal microscopy analysis of polarized or nonpolarized CAG myeloma cells following addition of CD63-RFP exosomes. Exosomes preferentially interact with heparan sulfate-rich uropods of polarized cells but are widely distributed on nonpolarized cells. B , depletion of heparan sulfate chains on myeloma cells decreases exosome-target cell interaction. RPMI-8226 cells were untreated or treated with heparitinase and washed, and their interactions with exosomes were analyzed by confocal microscopy. Right panel , quantitative data from a similar experiment analyzed by flow cytometry. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Exosome-target cell interaction is mediated by heparan sulfate chains on target cells. A , confocal microscopy analysis of polarized or nonpolarized CAG myeloma cells following addition of CD63-RFP exosomes. Exosomes preferentially interact with heparan sulfate-rich uropods of polarized cells but are widely distributed on nonpolarized cells. B , depletion of heparan sulfate chains on myeloma cells decreases exosome-target cell interaction. RPMI-8226 cells were untreated or treated with heparitinase and washed, and their interactions with exosomes were analyzed by confocal microscopy. Right panel , quantitative data from a similar experiment analyzed by flow cytometry. #, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Confocal Microscopy, Flow Cytometry, Cytometry

    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: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    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: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    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: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    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: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    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: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    Techniques: Affinity Precipitation, Stable Transfection, Expressing, Incubation

    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: Heparinase I ( Sigma Chemical Co. ) was used at 600 mU, and heparitinase II (Seikagaku Corporation, Tokyo, Japan) was used at 4 mU.

    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: Heparinase I ( Sigma Chemical Co. ) was used at 600 mU, and heparitinase II (Seikagaku Corporation, Tokyo, Japan) was used at 4 mU.

    Techniques: Immunofluorescence, Staining

    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay

    Heparanase acts through syntenin-1, ALIX and the syntenin-ALIX interaction to stimulate intraluminal budding. (A) The role of syntenin-1 and (B) the role of ALIX in the effect of heparanase on exosomes were investigated by the knockdown of syntenin-1 and ALIX (using Synt1 RNAi and ALIX RNAi, respectively) in MCF-7 cells. Non-targeting RNAi (NT) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both the experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that lysate and exosome samples derived from NT and KD cells, separated by a blank space in A , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (C) Confocal micrographs of MCF-7 cells co-transfected with wild-type mCherry-syntenin-1 (mCherry-Synt1 WT) or with mCherry-syntenin-1 harboring mutant LYP sequences and therefore defective in ALIX-binding (mCherry-Synt1-ΔALIX; red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge). Intraluminal budding of wild-type and mutant mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain was scored in the presence (Hep) or absence (no Hep) of heparanase. (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (blue in merge). In addition, the cells were transfected with RNAi targeting ALIX ( ALIX RNAi) or with non-targeting RNAi (NT RNAi). Intraluminal budding of mCherry-syntenin-1 was evaluated in the presence (Hep) or absence (no Hep) of heparanase. (E) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in C and (F) quantitation of intraluminal budding of mCherry-syntenin-1, as in D , measuring the mean fluorescence intensity of mCherry-syntenin-1 and syndecan-1 in the lumen of RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: Heparanase acts through syntenin-1, ALIX and the syntenin-ALIX interaction to stimulate intraluminal budding. (A) The role of syntenin-1 and (B) the role of ALIX in the effect of heparanase on exosomes were investigated by the knockdown of syntenin-1 and ALIX (using Synt1 RNAi and ALIX RNAi, respectively) in MCF-7 cells. Non-targeting RNAi (NT) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both the experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that lysate and exosome samples derived from NT and KD cells, separated by a blank space in A , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (C) Confocal micrographs of MCF-7 cells co-transfected with wild-type mCherry-syntenin-1 (mCherry-Synt1 WT) or with mCherry-syntenin-1 harboring mutant LYP sequences and therefore defective in ALIX-binding (mCherry-Synt1-ΔALIX; red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge). Intraluminal budding of wild-type and mutant mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain was scored in the presence (Hep) or absence (no Hep) of heparanase. (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (blue in merge). In addition, the cells were transfected with RNAi targeting ALIX ( ALIX RNAi) or with non-targeting RNAi (NT RNAi). Intraluminal budding of mCherry-syntenin-1 was evaluated in the presence (Hep) or absence (no Hep) of heparanase. (E) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in C and (F) quantitation of intraluminal budding of mCherry-syntenin-1, as in D , measuring the mean fluorescence intensity of mCherry-syntenin-1 and syndecan-1 in the lumen of RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay, Transfection, Mutagenesis, Binding Assay, Quantitation Assay, Fluorescence

    Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P

    Journal: Cell Research

    Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P

    Article Snippet: Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7).

    Techniques: Western Blot, Activity 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