heparitinase  (Millipore)


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    Structured Review

    Millipore heparitinase
    A component of N40 attachment to Vero cells is independent of GAGs. Vero cells were mock treated or were treated with the indicated lyases prior to the addition of radiolabeled strain N40. Hep., heparinase digestion; Hpt., <t>heparitinase</t> digestion; Chon. ABC, chondroitinase ABC digestion. Each bar represents the average ± the SD of four determinations. Significant ( P
    Heparitinase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/Millipore
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Strain Variation in Glycosaminoglycan Recognition Influences Cell-Type-Specific Binding by Lyme Disease Spirochetes"

    Article Title: Strain Variation in Glycosaminoglycan Recognition Influences Cell-Type-Specific Binding by Lyme Disease Spirochetes

    Journal: Infection and Immunity

    doi:

    A component of N40 attachment to Vero cells is independent of GAGs. Vero cells were mock treated or were treated with the indicated lyases prior to the addition of radiolabeled strain N40. Hep., heparinase digestion; Hpt., heparitinase digestion; Chon. ABC, chondroitinase ABC digestion. Each bar represents the average ± the SD of four determinations. Significant ( P
    Figure Legend Snippet: A component of N40 attachment to Vero cells is independent of GAGs. Vero cells were mock treated or were treated with the indicated lyases prior to the addition of radiolabeled strain N40. Hep., heparinase digestion; Hpt., heparitinase digestion; Chon. ABC, chondroitinase ABC digestion. Each bar represents the average ± the SD of four determinations. Significant ( P

    Techniques Used:

    2) Product Images from "The Brain Chondroitin Sulfate Proteoglycan Brevican Associates with Astrocytes Ensheathing Cerebellar Glomeruli and Inhibits Neurite Outgrowth from Granule Neurons"

    Article Title: The Brain Chondroitin Sulfate Proteoglycan Brevican Associates with Astrocytes Ensheathing Cerebellar Glomeruli and Inhibits Neurite Outgrowth from Granule Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.17-20-07784.1997

    A major portion of the brevican binding to B28 cells is independent of cell surface hyaluronan. A , Hyaluronidase treatment of B28 cells does not abolish the brevican binding to the cells. Monolayers of B28 cells were treated for 1 hr with hyaluronidase (10 and 20 TRU/ml), a mixture of heparinase (5 U/ml) and heparitinase (1 U/ml), and chondroitinase ABC (50 mU/ml). The binding assay was then performed with the ad dition of core proteins of the total soluble proteoglycan fraction, followed by incubations with RB18 and [ 125 I]anti-mouse IgG. Data represent means ± SD ( n = 3) of percent net binding relative to the binding to cells that were not treated with enzymes (defined as 100%). B , The 80 kDa brevican core protein lacking the hyaluronan-binding domain binds B28 cells. The binding assay was performed without addition (-), with addition of core proteins of the total soluble proteoglycan fraction ( CP ), or with addition of HPLC-purified 80 kDa brevican core protein ( 80K ). The amount of brevican bound to the cells was assayed with RB18 antibodies. Anti-chicken B-cadherin monoclonal antibody 5A6 was used as a negative control. Data represent means of duplicate determinations of net binding.
    Figure Legend Snippet: A major portion of the brevican binding to B28 cells is independent of cell surface hyaluronan. A , Hyaluronidase treatment of B28 cells does not abolish the brevican binding to the cells. Monolayers of B28 cells were treated for 1 hr with hyaluronidase (10 and 20 TRU/ml), a mixture of heparinase (5 U/ml) and heparitinase (1 U/ml), and chondroitinase ABC (50 mU/ml). The binding assay was then performed with the ad dition of core proteins of the total soluble proteoglycan fraction, followed by incubations with RB18 and [ 125 I]anti-mouse IgG. Data represent means ± SD ( n = 3) of percent net binding relative to the binding to cells that were not treated with enzymes (defined as 100%). B , The 80 kDa brevican core protein lacking the hyaluronan-binding domain binds B28 cells. The binding assay was performed without addition (-), with addition of core proteins of the total soluble proteoglycan fraction ( CP ), or with addition of HPLC-purified 80 kDa brevican core protein ( 80K ). The amount of brevican bound to the cells was assayed with RB18 antibodies. Anti-chicken B-cadherin monoclonal antibody 5A6 was used as a negative control. Data represent means of duplicate determinations of net binding.

    Techniques Used: Binding Assay, High Performance Liquid Chromatography, Purification, Negative Control

    3) Product Images from "Osteoblastic heparan sulfate regulates osteoprotegerin function and bone mass"

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.89624

    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
    Figure Legend 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

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

    4) Product Images from "Agrin Can Mediate Acetylcholine Receptor Gene Expression in Muscle by Aggregation of Muscle-derived Neuregulins "

    Article Title: Agrin Can Mediate Acetylcholine Receptor Gene Expression in Muscle by Aggregation of Muscle-derived Neuregulins

    Journal: The Journal of Cell Biology

    doi:

    Binding of HRGs to agrin is mediated by GAG chains at the NH 2 -terminal part of agrin and requires the Ig-like domain of NRGs. ( A ) Transfer blot overlay assay of native full-length cAgrin 7A0B0 ( Heparitinase −) or enzyme-treated cAgrin 7A0B0 ( Heparitinase +) with HRGγ ( OL: HRGγ ). Treatment with heparitinase causes a shift in the molecular weight of full-length agrin as indicated by immunoblot analysis using anti-agrin antibodies ( anti-Agrin ) and strongly reduces binding of HRGγ. ( B ) HRGγ binds to full-length agrin isoforms but not to COOH-terminal agrin fragments. Solid phase radioligand-binding assay of 125 I-HRGγ to immobilized full-length agrin isoforms, Agrin-c95 A0B0 and BSA. Mean values ± SEM ( n = 3) of bound 125 I-HRGγ (in cpm). ( C ) The Ig-like domain of HRGs is sufficient to bind to full-length agrin. Transfer blot overlay assays ( OL ) of Agrin 7A4B8 and Agrin 7A0B0 with HRGΔBbsI in the presence (+) or absence (−) of 10 mM β-mercaptoethanol ( β-ME ) and detection with anti-His primary antibody ( arrow ). The Ig domain of HRGs binds to immobilized agrin even under reducing conditions, indicating that the disulfide bond of the HRG Ig domain might not be critical for interaction with agrin. In contrast, no binding is detected in overlay assays with HRGβ1 (177–246) and anti–FLAG M1 primary antibody. Schematic representations of the HRG fragments used in this overlay experiment are indicated.
    Figure Legend Snippet: Binding of HRGs to agrin is mediated by GAG chains at the NH 2 -terminal part of agrin and requires the Ig-like domain of NRGs. ( A ) Transfer blot overlay assay of native full-length cAgrin 7A0B0 ( Heparitinase −) or enzyme-treated cAgrin 7A0B0 ( Heparitinase +) with HRGγ ( OL: HRGγ ). Treatment with heparitinase causes a shift in the molecular weight of full-length agrin as indicated by immunoblot analysis using anti-agrin antibodies ( anti-Agrin ) and strongly reduces binding of HRGγ. ( B ) HRGγ binds to full-length agrin isoforms but not to COOH-terminal agrin fragments. Solid phase radioligand-binding assay of 125 I-HRGγ to immobilized full-length agrin isoforms, Agrin-c95 A0B0 and BSA. Mean values ± SEM ( n = 3) of bound 125 I-HRGγ (in cpm). ( C ) The Ig-like domain of HRGs is sufficient to bind to full-length agrin. Transfer blot overlay assays ( OL ) of Agrin 7A4B8 and Agrin 7A0B0 with HRGΔBbsI in the presence (+) or absence (−) of 10 mM β-mercaptoethanol ( β-ME ) and detection with anti-His primary antibody ( arrow ). The Ig domain of HRGs binds to immobilized agrin even under reducing conditions, indicating that the disulfide bond of the HRG Ig domain might not be critical for interaction with agrin. In contrast, no binding is detected in overlay assays with HRGβ1 (177–246) and anti–FLAG M1 primary antibody. Schematic representations of the HRG fragments used in this overlay experiment are indicated.

    Techniques Used: Binding Assay, Overlay Assay, Molecular Weight, Radio Ligand Binding Assay

    5) Product Images from "The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification"

    Article Title: The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

    Journal: Molecular and Cellular Biology

    doi:

    Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.
    Figure Legend Snippet: Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Techniques Used: Modification, Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation

    6) Product Images from "Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation"

    Article Title: Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-04-01226.1999

    Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p
    Figure Legend Snippet: Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Techniques Used:

    7) Product Images from "Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor"

    Article Title: Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    Journal: Molecular Biology of the Cell

    doi:

    Histochemical staining of tumors derived from pancreata of RIP-Tag2 mice. Cryostat sections of mouse pancreas tumors were reacted with chimeras (0.5 μg/ml) and processed for histochemistry as described in MATERIALS AND METHODS. Original magnification 50× in A and 400× in B–E. (A) Control fusion protein (E-cadherin IgG) staining of tumor section. The ECM lacks staining. (B) Layilin-IgG stains positively the ECM, and the staining is sensitive to hyaluronidase treatment before incubation with layilin-IgG (C). Similar pretreatment of sections with chondroitinase (D) or heparitinase (E) did not abolish layilin-Fc reactivity.
    Figure Legend Snippet: Histochemical staining of tumors derived from pancreata of RIP-Tag2 mice. Cryostat sections of mouse pancreas tumors were reacted with chimeras (0.5 μg/ml) and processed for histochemistry as described in MATERIALS AND METHODS. Original magnification 50× in A and 400× in B–E. (A) Control fusion protein (E-cadherin IgG) staining of tumor section. The ECM lacks staining. (B) Layilin-IgG stains positively the ECM, and the staining is sensitive to hyaluronidase treatment before incubation with layilin-IgG (C). Similar pretreatment of sections with chondroitinase (D) or heparitinase (E) did not abolish layilin-Fc reactivity.

    Techniques Used: Staining, Derivative Assay, Mouse Assay, Incubation

    8) Product Images from "Different Classes of Proteoglycans Contribute to the Attachment of Borrelia burgdorferi to Cultured Endothelial and Brain Cells"

    Article Title: Different Classes of Proteoglycans Contribute to the Attachment of Borrelia burgdorferi to Cultured Endothelial and Brain Cells

    Journal: Infection and Immunity

    doi:

    Attachment of B. burgdorferi to Vero cells is sensitive to heparinase or heparitinase digestion, while attachment to 293 cells is sensitive to chondroitinase ABC digestion. Bacterial attachment of the infectious B. burgdorferi strain N40, clone D10/E9, to confluent monolayers was determined as described in Materials and Methods. (A) Attachment of N40 to Vero cells or 293 cells was determined (from left to right) in the absence of inhibitor or in the presence of 500 μg of chondroitin-6-sulfate, chondroitin-4-sulfate, heparin, heparan sulfate, or dermatan sulfate per ml. (B) Bacterial attachment was quantitated (from left to right) after no treatment or after a 2-h treatment of the monolayers with 0.5 U of heparinase, heparitinase, or chondroitinase ABC per ml.
    Figure Legend Snippet: Attachment of B. burgdorferi to Vero cells is sensitive to heparinase or heparitinase digestion, while attachment to 293 cells is sensitive to chondroitinase ABC digestion. Bacterial attachment of the infectious B. burgdorferi strain N40, clone D10/E9, to confluent monolayers was determined as described in Materials and Methods. (A) Attachment of N40 to Vero cells or 293 cells was determined (from left to right) in the absence of inhibitor or in the presence of 500 μg of chondroitin-6-sulfate, chondroitin-4-sulfate, heparin, heparan sulfate, or dermatan sulfate per ml. (B) Bacterial attachment was quantitated (from left to right) after no treatment or after a 2-h treatment of the monolayers with 0.5 U of heparinase, heparitinase, or chondroitinase ABC per ml.

    Techniques Used:

    Related Articles

    Centrifugation:

    Article Title: Virion-associated viral fibroblast growth factor stimulates cell motility
    Article Snippet: .. To remove virion-bound vFGF, 3 × 107 AcBAC-HSP70vFGFHA virions in 3 ml of buffer (20 mM Tris-HCl, pH 7, 0.1mg/ml BSA, and 4 mM CaCl2 ) were treated with 1IU of heparinase III from Flavobacterium heparinum (Sigma Aldrich) at 27 °C for 4 h. After treatment, virions were purified by centrifugation at 24,000 × g through a 25% Nycoprep cushion. ..

    Labeling:

    Article Title: Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum
    Article Snippet: .. Human bloodgroup O Rh+ erythrocytes (5% suspension in RPMI) were, before C-FDA labeling , incubated for 60–90 min with either heparinase III (25°C, pH 7.5; Sigma Chemical Co. ), chondroitinase ABC (37°C, pH 8.0; Sigma Chemical Co. ) or with clostridium perfringens neuraminidase (37°C, pH 6.0; Sigma Chemical Co. ). ..

    Purification:

    Article Title: Virion-associated viral fibroblast growth factor stimulates cell motility
    Article Snippet: .. To remove virion-bound vFGF, 3 × 107 AcBAC-HSP70vFGFHA virions in 3 ml of buffer (20 mM Tris-HCl, pH 7, 0.1mg/ml BSA, and 4 mM CaCl2 ) were treated with 1IU of heparinase III from Flavobacterium heparinum (Sigma Aldrich) at 27 °C for 4 h. After treatment, virions were purified by centrifugation at 24,000 × g through a 25% Nycoprep cushion. ..

    Incubation:

    Article Title: Dendritic space-filling requires a neuronal type-specific extracellular permissive signal in Drosophila
    Article Snippet: .. Briefly, larval fillets were incubated with 500 mU/mL heparinase III (Sigma) in 50 mM Tris·HCl (pH 7.2), 100 mM NaCl, 1 mM CaCl2 , 0.1% Triton-X100, 5 mg/mL BSA for 6 h at 37 °C. .. Then, larval fillets were stained with primary (F69-3G10) and secondary antibodies.

    Article Title: Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum
    Article Snippet: .. Human bloodgroup O Rh+ erythrocytes (5% suspension in RPMI) were, before C-FDA labeling , incubated for 60–90 min with either heparinase III (25°C, pH 7.5; Sigma Chemical Co. ), chondroitinase ABC (37°C, pH 8.0; Sigma Chemical Co. ) or with clostridium perfringens neuraminidase (37°C, pH 6.0; Sigma Chemical Co. ). ..

    other:

    Article Title: Combinatorial Targeting of the Macropinocytotic Pathway in Leukemia and Lymphoma Cells *
    Article Snippet: Reagents —Methyl β -cyclodextrin, chlorpromazine, wortmannin, 5-( N -ethyl- N -isopropyl)amiloride, propidium iodide, sodium heparin, heparinase III, fluorescein isothiocyanate-labeled transferrin (FITC-Tf), and rabbit anti-fd bacteriophage antibody were purchased from Sigma.

    Activity Assay:

    Article Title: A Biomechanical Role for Perlecan in the Pericellular Matrix of Articular Cartilage
    Article Snippet: .. Heparinase III (heparitinase I; EC 4.2.2.8; Sigma-Aldrich, Inc., St. Louis, MO) is the most specific heparinase for HS, demonstrating no activity for heparin ( ). .. Cartilage sections were incubated in 50 μL of 6 U/mL (0.01 IU/mL) heparinase III solution in 20 mM Tris-HCl containing 0.1 mg/mL bovine serum albumin (BSA) and 4 mM CaCl2 , pH 7.0 at 37°C for 30 minutes.

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    Millipore heparitinase
    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of <t>heparitinase</t> treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P
    Heparitinase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/Millipore
    Average 99 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    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

    Heparin competes with Wnt5A binding of HS, and removal of HS decreases motility in melanoma cell lines. Cells were treated with increasing doses of heparin, and the medium of the cells was examined for Wnt5A release. A , in the presence of heparin, Wnt5A accumulates in the medium. The addition of rWnt5A to cells in the absence of heparin results in its rapid uptake and internalization, but in the presence of heparin, rWnt5A just accumulates in the medium. B , PKC signaling is affected by high doses of heparin, regardless of whether rWnt5A is added. C , cells were treated with 2 milliunits/ml heparinase III for 24 h and examined for PKC signaling. PKC signaling is decreased upon heparinase III treatment and partially reconstituted upon the addition of rWnt5A. D , representative images of the motility assays using M93-047 cells demonstrating that heparinase III treatment decreased the rate of wound closure. E , graphical representation of UACC903 scratch closure rates demonstrating that rWnt5A addition can overcome heparinase III treatment. F , quantitative extracellular matrix invasion assays also demonstrate that heparinase III treatment decreases the rate of melanoma cell invasion, and this can be restored upon the addition of exogenous Wnt5A to the media of the cells ( n = 3; error bars show S.D.; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells *

    doi: 10.1074/jbc.M109.028498

    Figure Lengend Snippet: Heparin competes with Wnt5A binding of HS, and removal of HS decreases motility in melanoma cell lines. Cells were treated with increasing doses of heparin, and the medium of the cells was examined for Wnt5A release. A , in the presence of heparin, Wnt5A accumulates in the medium. The addition of rWnt5A to cells in the absence of heparin results in its rapid uptake and internalization, but in the presence of heparin, rWnt5A just accumulates in the medium. B , PKC signaling is affected by high doses of heparin, regardless of whether rWnt5A is added. C , cells were treated with 2 milliunits/ml heparinase III for 24 h and examined for PKC signaling. PKC signaling is decreased upon heparinase III treatment and partially reconstituted upon the addition of rWnt5A. D , representative images of the motility assays using M93-047 cells demonstrating that heparinase III treatment decreased the rate of wound closure. E , graphical representation of UACC903 scratch closure rates demonstrating that rWnt5A addition can overcome heparinase III treatment. F , quantitative extracellular matrix invasion assays also demonstrate that heparinase III treatment decreases the rate of melanoma cell invasion, and this can be restored upon the addition of exogenous Wnt5A to the media of the cells ( n = 3; error bars show S.D.; **, p

    Article Snippet: Cells were serum-starved for 2 h and treated with 2 milliunits/ml heparinase III for 24 h and/or rWnt5A or rWnt3A for 16 h. Cells were then seeded at 5 × 105 cells/ml in the heparinase III/Wnt5A serum-free media in the transwell insert of a QCM plate (Millipore, Billerica, MA).

    Techniques: Binding Assay

    Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Journal: Molecular and Cellular Biology

    Article Title: The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

    doi:

    Figure Lengend Snippet: Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Article Snippet: Cell lysates from affinity-labeling samples were diluted at least 50-fold in a 0.1 M Tris acetate buffer and treated with heparitinase (code 100703; lot no. E95601; Seikagaku America, Rockville, Md.) and/or chondroitinase ABC (code 100332; lot no. KE95702; Seikagaku America) (final concentration, 10 mIU/ml) in 0.1 M Tris acetate buffer, pH 7.3, at 37°C for 1 h. In the case of treatment with heparitinase alone, shark cartilage chondroitin sulfate (catalog no. 2307; Calbiochem) was added to a final concentration of 50 μg/ml to protect the samples from digestion by chondroitinases possibly contaminating the heparitinase preparation.

    Techniques: Modification, Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation

    Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Journal: The Journal of Neuroscience

    Article Title: Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation

    doi: 10.1523/JNEUROSCI.19-04-01226.1999

    Figure Lengend Snippet: Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Article Snippet: Aliquots of the fractions were digested with nitrous acid or heparitinase (10 U/ml, 15 hr) to remove carbohydrate side chains or incubated with 50 μ m herbimycin A (Calbiochem, La Jolla, CA).

    Techniques: