Structured Review

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 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Expression of c-Met and Heparan-Sulfate Proteoglycan Forms of CD44 in Colorectal Cancer"

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

Journal: The American Journal of Pathology

doi:

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.
Figure Legend 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.

Techniques Used: 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 .
Figure Legend 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 .

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

2) Product Images from "Glycosaminoglycans in human retinoblastoma cells: Heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions"

Article Title: Glycosaminoglycans in human retinoblastoma cells: Heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-4-1

Effect of GAG lyases and chlorate on 125 I-PEDF binding to retinoblastoma Y-79 cells . Human retinoblastoma Y-79 cells (5 × 10 5 cells/ml) were cultured in serum-free media at 37°C and treated with GAG lyases or chlorate. Binding to the treated cells was performed with 2 nM 125 I-PEDF. Free and bound PEDF were separated by filtration through glass-fiber filters under vacuum. Non-specific binding was determined from binding reactions in the presence of 50-fold excess of unlabeled ligand. Specific binding was calculated by subtracting non-specific binding from total binding. Panel A. Cells cultured for 16 h were treated with hyaluronidase, heparinase and heparitinase at 37°C for 120 min. All experimental points are given as the average of quadruplicates. Panel B. Cells were cultured with or without 30 mM sodium chlorate and 10 mM sodium sulfate at 37°C for 16 h before the radioligand was added. The binding reactions were at 4°C for 30 min. All experimental points are given as the average of triplicates.
Figure Legend Snippet: Effect of GAG lyases and chlorate on 125 I-PEDF binding to retinoblastoma Y-79 cells . Human retinoblastoma Y-79 cells (5 × 10 5 cells/ml) were cultured in serum-free media at 37°C and treated with GAG lyases or chlorate. Binding to the treated cells was performed with 2 nM 125 I-PEDF. Free and bound PEDF were separated by filtration through glass-fiber filters under vacuum. Non-specific binding was determined from binding reactions in the presence of 50-fold excess of unlabeled ligand. Specific binding was calculated by subtracting non-specific binding from total binding. Panel A. Cells cultured for 16 h were treated with hyaluronidase, heparinase and heparitinase at 37°C for 120 min. All experimental points are given as the average of quadruplicates. Panel B. Cells were cultured with or without 30 mM sodium chlorate and 10 mM sodium sulfate at 37°C for 16 h before the radioligand was added. The binding reactions were at 4°C for 30 min. All experimental points are given as the average of triplicates.

Techniques Used: Binding Assay, Cell Culture, Filtration

Spectrophotometric assays for heparin and HS-like molecules . Heparin and HS were degraded with heparinase and heparitinase, respectively. Proteins in CM concentrated 10-fold and CM PEDF were digested first with subtilisin. Protease-treated CM and CM PEDF were reacted with heparinase and heparitinase. The appearance of degradation products (Δ 4 -hexuronate) was measured by absorbance at 235 nm (Extinction coefficient = 5500). Panels A and B show heparinase activity and panels C and D show heparitinase activity. Time course and concentration curves of enzymes using their respective substrates are shown in panels A and C. Panels B and D correspond to reactions with CM and CM PEDF .
Figure Legend Snippet: Spectrophotometric assays for heparin and HS-like molecules . Heparin and HS were degraded with heparinase and heparitinase, respectively. Proteins in CM concentrated 10-fold and CM PEDF were digested first with subtilisin. Protease-treated CM and CM PEDF were reacted with heparinase and heparitinase. The appearance of degradation products (Δ 4 -hexuronate) was measured by absorbance at 235 nm (Extinction coefficient = 5500). Panels A and B show heparinase activity and panels C and D show heparitinase activity. Time course and concentration curves of enzymes using their respective substrates are shown in panels A and C. Panels B and D correspond to reactions with CM and CM PEDF .

Techniques Used: Activity Assay, Concentration Assay

Related Articles

Avidin-Biotin Assay:

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

Expressing:

Article Title: Regulation of Cytokine Signaling by B Cell Antigen Receptor and Cd40-Controlled Expression of Heparan Sulfate Proteoglycans
Article Snippet: .. For enzymatic cleavage of HS, cells or tissue sections were treated with 10 mU/ml heparitinase (Flavobacterium heparinum , EC 4.2.2.8; ICN Biomedicals) in RPMI 1640 (GIBCO BRL/Life Technologies) at 37°C for 3 h. The cleavage of HS by heparitinase was determined by the loss of cell surface–expressed HS (mAb 10E4), and the simultaneous gain of HS-stub expression (mAb 3G10). ..

Purification:

Article Title: Glycosaminoglycans in human retinoblastoma cells: Heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions
Article Snippet: .. Heparitinase (E.C.4.2.2.8) and heparinase (E.C.4.2.2.7) purified from Flavobacterium heparinum were from ICN Biomedicals, Inc. and alternatively from Seikagaku. .. Heparan sulfate (HS) purified from bovine kidney was from Seikagaku, hyaluronidase (E.C.4.2.2.1) purified from Streptomyces hyalurolyticus from ICN Pharmaceuticals, Coomassie Brilliant Blue from BioRad, and Q-Sepharose from Pharmacia.

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    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 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/ICN Biomedicals
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    85
    ICN Biomedicals heparitinase buffer
    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 Buffer, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase buffer/product/ICN Biomedicals
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    heparitinase buffer - by Bioz Stars, 2020-11
    85/100 stars
      Buy from Supplier

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    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

    Effect of GAG lyases and chlorate on 125 I-PEDF binding to retinoblastoma Y-79 cells . Human retinoblastoma Y-79 cells (5 × 10 5 cells/ml) were cultured in serum-free media at 37°C and treated with GAG lyases or chlorate. Binding to the treated cells was performed with 2 nM 125 I-PEDF. Free and bound PEDF were separated by filtration through glass-fiber filters under vacuum. Non-specific binding was determined from binding reactions in the presence of 50-fold excess of unlabeled ligand. Specific binding was calculated by subtracting non-specific binding from total binding. Panel A. Cells cultured for 16 h were treated with hyaluronidase, heparinase and heparitinase at 37°C for 120 min. All experimental points are given as the average of quadruplicates. Panel B. Cells were cultured with or without 30 mM sodium chlorate and 10 mM sodium sulfate at 37°C for 16 h before the radioligand was added. The binding reactions were at 4°C for 30 min. All experimental points are given as the average of triplicates.

    Journal: BMC Biochemistry

    Article Title: Glycosaminoglycans in human retinoblastoma cells: Heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions

    doi: 10.1186/1471-2091-4-1

    Figure Lengend Snippet: Effect of GAG lyases and chlorate on 125 I-PEDF binding to retinoblastoma Y-79 cells . Human retinoblastoma Y-79 cells (5 × 10 5 cells/ml) were cultured in serum-free media at 37°C and treated with GAG lyases or chlorate. Binding to the treated cells was performed with 2 nM 125 I-PEDF. Free and bound PEDF were separated by filtration through glass-fiber filters under vacuum. Non-specific binding was determined from binding reactions in the presence of 50-fold excess of unlabeled ligand. Specific binding was calculated by subtracting non-specific binding from total binding. Panel A. Cells cultured for 16 h were treated with hyaluronidase, heparinase and heparitinase at 37°C for 120 min. All experimental points are given as the average of quadruplicates. Panel B. Cells were cultured with or without 30 mM sodium chlorate and 10 mM sodium sulfate at 37°C for 16 h before the radioligand was added. The binding reactions were at 4°C for 30 min. All experimental points are given as the average of triplicates.

    Article Snippet: Heparitinase (E.C.4.2.2.8) and heparinase (E.C.4.2.2.7) purified from Flavobacterium heparinum were from ICN Biomedicals, Inc. and alternatively from Seikagaku.

    Techniques: Binding Assay, Cell Culture, Filtration

    Spectrophotometric assays for heparin and HS-like molecules . Heparin and HS were degraded with heparinase and heparitinase, respectively. Proteins in CM concentrated 10-fold and CM PEDF were digested first with subtilisin. Protease-treated CM and CM PEDF were reacted with heparinase and heparitinase. The appearance of degradation products (Δ 4 -hexuronate) was measured by absorbance at 235 nm (Extinction coefficient = 5500). Panels A and B show heparinase activity and panels C and D show heparitinase activity. Time course and concentration curves of enzymes using their respective substrates are shown in panels A and C. Panels B and D correspond to reactions with CM and CM PEDF .

    Journal: BMC Biochemistry

    Article Title: Glycosaminoglycans in human retinoblastoma cells: Heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions

    doi: 10.1186/1471-2091-4-1

    Figure Lengend Snippet: Spectrophotometric assays for heparin and HS-like molecules . Heparin and HS were degraded with heparinase and heparitinase, respectively. Proteins in CM concentrated 10-fold and CM PEDF were digested first with subtilisin. Protease-treated CM and CM PEDF were reacted with heparinase and heparitinase. The appearance of degradation products (Δ 4 -hexuronate) was measured by absorbance at 235 nm (Extinction coefficient = 5500). Panels A and B show heparinase activity and panels C and D show heparitinase activity. Time course and concentration curves of enzymes using their respective substrates are shown in panels A and C. Panels B and D correspond to reactions with CM and CM PEDF .

    Article Snippet: Heparitinase (E.C.4.2.2.8) and heparinase (E.C.4.2.2.7) purified from Flavobacterium heparinum were from ICN Biomedicals, Inc. and alternatively from Seikagaku.

    Techniques: Activity Assay, Concentration Assay