keratanase Search Results


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  • 94
    Millipore keratanase
    Western blots depicting the expression of decorin and biglycan after proteoglycan extraction from non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP). For each Western blot, lane (a) is undigested (U) proteoglycan extract from NCD (mongrel) and lane (c) is from CD (beagle) canine NP. Lanes (b) and (d) depict the detection of the specific small leucine-rich proteoglycan (SLRP) indicated above after digestion (D) with chondroitinase ABC and <t>keratinase</t> in NCD canine ( lane b ) and CD canine ( lane d ). a The decorin core protein is visualized appropriately at the 43 kDa molecular weight after digestion ( lane b ), with a clear, single band found from NCD NP extracts. NP extracts from CD canines ( lane d ) reveal fragmentation of decorin with multiple lower molecular weight bands visualized at 30, 25–20, and 17–15 kDa. b The biglycan core protein provides a clear, single band at the 45 kDa molecular weight in the NCD NP extract; however, the CD NP extract reveals multiple fragments of the core protein visualized at 37–35, 28, and 25 kDa
    Keratanase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase/product/Millipore
    Average 94 stars, based on 122 article reviews
    Price from $9.99 to $1999.99
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    90
    Seikagaku keratanase ii
    SDS-PAGE and immunoblotting of aggrecan with antibodies to different regions. Proteoglycan from guanidine extracts of OA articular cartilage, treated with <t>keratanase</t> and chondroitinase ABC, was analyzed by electrophoresis on polyacrylamide gels, and the fractionated proteoglycan then transferred to nitrocellulose membranes. Aggrecan was visualized by immunoblotting using antibodies recognizing the G1, G2, CS1 or G3 regions. Cartilage samples were obtained adjacent to the lesion (L), midway between the lesion and the joint margin (M), and remote from the lesion (R) from the femoral condyles. Representative samples from patient 1 are shown. Molecular weights of reference proteins are indicated at the left hand side of the blot. Schematic diagrams depicting the core protein structure of aggrecan fragments of different sizes are included at the right side of the blot
    Keratanase Ii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 90/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase ii/product/Seikagaku
    Average 90 stars, based on 248 article reviews
    Price from $9.99 to $1999.99
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    90/100 stars
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    90
    Seikagaku keratanase
    Western analysis demonstrated that <t>keratanase</t> reduces the phosphorylation of an 80 kDa protein. An anti-phosphotyrosine antibody was used to examine whether keratanase altered the phosphorylation of specific proteins in dentate explants. In control explants treated with HBSS, a strong band was present at ∼80 kDa. This band was not detectable in homogenates from cultures treated with keratanases I+II (KIK2).
    Keratanase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 90/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase/product/Seikagaku
    Average 90 stars, based on 232 article reviews
    Price from $9.99 to $1999.99
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    92
    AMS Biotechnology keratanase
    Western analysis demonstrated that <t>keratanase</t> reduces the phosphorylation of an 80 kDa protein. An anti-phosphotyrosine antibody was used to examine whether keratanase altered the phosphorylation of specific proteins in dentate explants. In control explants treated with HBSS, a strong band was present at ∼80 kDa. This band was not detectable in homogenates from cultures treated with keratanases I+II (KIK2).
    Keratanase, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase/product/AMS Biotechnology
    Average 92 stars, based on 9 article reviews
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    94
    Millipore keratanase i
    Western analysis demonstrated that <t>keratanase</t> reduces the phosphorylation of an 80 kDa protein. An anti-phosphotyrosine antibody was used to examine whether keratanase altered the phosphorylation of specific proteins in dentate explants. In control explants treated with HBSS, a strong band was present at ∼80 kDa. This band was not detectable in homogenates from cultures treated with keratanases I+II (KIK2).
    Keratanase I, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase i/product/Millipore
    Average 94 stars, based on 19 article reviews
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    88
    Seikagaku keratanase i
    ADAMTS12 is an evolutionarily conserved chondroitin sulfate/dermatan sulfate proteoglycan. ( A ) Schematic of ADAMTS12 domain structure. TSR, thrombospondin type 1 repeat; CS, chondroitin sulfate. ( B ) Alignment of human and mouse ADAMTS12 mucin modules showing a high degree of conservation (asterisks show identical residues, residue numbers are shown on the right), potential N-glycosylation sites (bold) and Ser-Gly dipeptide motifs (red) with acidic neighbors, constituting putative glycosaminoglycan attachment sites. ( C ) Sequence of the corresponding module from the Ciona intestinalis ortholog of ADAMTS7 and ADAMTS12. Sequence motifs for N-glycosylation (bold) and Ser-Gly/Gly-Ser dipeptides (red) are shown. ( D ) Representative Western blot (anti-FLAG) of conditioned medium from transiently transfected HEK293F cells shows increased ADAMTS12 band intensity (arrows) following digestion with chondroitinase ABC (C’ABC), but not heparanase or <t>keratanase</t> I II. The blot is representative of n = 3. ( E ) Mutation of Ser 1157 and Ser 1159 (boxed residues in panel B ), but not Ser 1157 alone eliminates enhanced signal after C’ABC digestion and reactivity to an anti–chondroitin sulfate stub antibody (MAB2035) strongly suggestive of chondroitin/dermatan sulfate attachment at Ser 1157 and Ser 1159 . EV, empty vector; WT, wild type.
    Keratanase I, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase i/product/Seikagaku
    Average 88 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
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    90
    Valiant keratanase
    ADAMTS12 is an evolutionarily conserved chondroitin sulfate/dermatan sulfate proteoglycan. ( A ) Schematic of ADAMTS12 domain structure. TSR, thrombospondin type 1 repeat; CS, chondroitin sulfate. ( B ) Alignment of human and mouse ADAMTS12 mucin modules showing a high degree of conservation (asterisks show identical residues, residue numbers are shown on the right), potential N-glycosylation sites (bold) and Ser-Gly dipeptide motifs (red) with acidic neighbors, constituting putative glycosaminoglycan attachment sites. ( C ) Sequence of the corresponding module from the Ciona intestinalis ortholog of ADAMTS7 and ADAMTS12. Sequence motifs for N-glycosylation (bold) and Ser-Gly/Gly-Ser dipeptides (red) are shown. ( D ) Representative Western blot (anti-FLAG) of conditioned medium from transiently transfected HEK293F cells shows increased ADAMTS12 band intensity (arrows) following digestion with chondroitinase ABC (C’ABC), but not heparanase or <t>keratanase</t> I II. The blot is representative of n = 3. ( E ) Mutation of Ser 1157 and Ser 1159 (boxed residues in panel B ), but not Ser 1157 alone eliminates enhanced signal after C’ABC digestion and reactivity to an anti–chondroitin sulfate stub antibody (MAB2035) strongly suggestive of chondroitin/dermatan sulfate attachment at Ser 1157 and Ser 1159 . EV, empty vector; WT, wild type.
    Keratanase, supplied by Valiant, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase/product/Valiant
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    90/100 stars
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    91
    Glaxo Smith keratanase ii
    ADAMTS12 is an evolutionarily conserved chondroitin sulfate/dermatan sulfate proteoglycan. ( A ) Schematic of ADAMTS12 domain structure. TSR, thrombospondin type 1 repeat; CS, chondroitin sulfate. ( B ) Alignment of human and mouse ADAMTS12 mucin modules showing a high degree of conservation (asterisks show identical residues, residue numbers are shown on the right), potential N-glycosylation sites (bold) and Ser-Gly dipeptide motifs (red) with acidic neighbors, constituting putative glycosaminoglycan attachment sites. ( C ) Sequence of the corresponding module from the Ciona intestinalis ortholog of ADAMTS7 and ADAMTS12. Sequence motifs for N-glycosylation (bold) and Ser-Gly/Gly-Ser dipeptides (red) are shown. ( D ) Representative Western blot (anti-FLAG) of conditioned medium from transiently transfected HEK293F cells shows increased ADAMTS12 band intensity (arrows) following digestion with chondroitinase ABC (C’ABC), but not heparanase or <t>keratanase</t> I II. The blot is representative of n = 3. ( E ) Mutation of Ser 1157 and Ser 1159 (boxed residues in panel B ), but not Ser 1157 alone eliminates enhanced signal after C’ABC digestion and reactivity to an anti–chondroitin sulfate stub antibody (MAB2035) strongly suggestive of chondroitin/dermatan sulfate attachment at Ser 1157 and Ser 1159 . EV, empty vector; WT, wild type.
    Keratanase Ii, supplied by Glaxo Smith, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase ii/product/Glaxo Smith
    Average 91 stars, based on 1 article reviews
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    90
    Boehringer Mannheim keratanase
    Synthesis of osteoadherin by primary bovine osteoblasts and digestion of the immunoprecipitated osteoadherin with <t>keratanase</t> and N -glycosidase F. Osteoblasts were prepared from bovine bone using the method of Robey ( 41 ) and grown to confluency in Ham's F12 medium containing 10% calf serum. The cells were metabolically labeled in the presence of [ 35 S]sulfate for 6 h. Radiolabeled proteins in the culture medium were immunoprecipitated with the osteoadherin antiserum. The precipitates were digested with two different glycosidases. Digested samples, undigested control, and the whole cell medium were electrophoresed on 4–16% gradient polyacrylamide gels and stained with Coomassie blue. Labeled proteins were detected by fluorography. ( A ) Immunoprecipitated osteoadherin in the left lane, immunoprecipitated osteoadherin digested with keratanase in the middle lane, and nonprecipitated whole culture medium in the right lane. ( B ) Immunoprecipitated osteoadherin in the left lane, and immunoprecipitated osteoadherin digested with N -glycosidase F (N-glycanase) in the right lane. Both digestions were performed according to the recommendations of the manufacturers. The positions of molecular mass markers are indicated by arrows.
    Keratanase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase/product/Boehringer Mannheim
    Average 90 stars, based on 6 article reviews
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    90
    ICN Biomedicals keratanase
    Immunoblot analysis of purified proteoglycan with monoclonal antibodies TG343 and GCTM-2. Purified proteoglycan in its native form (N), or following treatment with chondroitinase ABC (C) or <t>keratanase</t> (K), was electrophoresed on a 7.5% SDS-PAGE gel under reducing conditions, transferred to Immobilon membrane, and probed with antibody TG343 (left) or GCTM-2 (right). The position of the 205-kDa molecular weight marker is indicated by the arrow.
    Keratanase, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanase/product/ICN Biomedicals
    Average 90 stars, based on 3 article reviews
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    85
    Seikagaku keratanases
    Immunoblot analysis of purified proteoglycan with monoclonal antibodies TG343 and GCTM-2. Purified proteoglycan in its native form (N), or following treatment with chondroitinase ABC (C) or <t>keratanase</t> (K), was electrophoresed on a 7.5% SDS-PAGE gel under reducing conditions, transferred to Immobilon membrane, and probed with antibody TG343 (left) or GCTM-2 (right). The position of the 205-kDa molecular weight marker is indicated by the arrow.
    Keratanases, supplied by Seikagaku, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratanases/product/Seikagaku
    Average 85 stars, based on 18 article reviews
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    84
    GenScript oligonucleotide encoding bc keratanase ii
    Immunoblot analysis of purified proteoglycan with monoclonal antibodies TG343 and GCTM-2. Purified proteoglycan in its native form (N), or following treatment with chondroitinase ABC (C) or <t>keratanase</t> (K), was electrophoresed on a 7.5% SDS-PAGE gel under reducing conditions, transferred to Immobilon membrane, and probed with antibody TG343 (left) or GCTM-2 (right). The position of the 205-kDa molecular weight marker is indicated by the arrow.
    Oligonucleotide Encoding Bc Keratanase Ii, supplied by GenScript, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligonucleotide encoding bc keratanase ii/product/GenScript
    Average 84 stars, based on 1 article reviews
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    Image Search Results


    Western blots depicting the expression of decorin and biglycan after proteoglycan extraction from non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP). For each Western blot, lane (a) is undigested (U) proteoglycan extract from NCD (mongrel) and lane (c) is from CD (beagle) canine NP. Lanes (b) and (d) depict the detection of the specific small leucine-rich proteoglycan (SLRP) indicated above after digestion (D) with chondroitinase ABC and keratinase in NCD canine ( lane b ) and CD canine ( lane d ). a The decorin core protein is visualized appropriately at the 43 kDa molecular weight after digestion ( lane b ), with a clear, single band found from NCD NP extracts. NP extracts from CD canines ( lane d ) reveal fragmentation of decorin with multiple lower molecular weight bands visualized at 30, 25–20, and 17–15 kDa. b The biglycan core protein provides a clear, single band at the 45 kDa molecular weight in the NCD NP extract; however, the CD NP extract reveals multiple fragments of the core protein visualized at 37–35, 28, and 25 kDa

    Journal: Arthritis Research & Therapy

    Article Title: The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc

    doi: 10.1186/s13075-015-0733-z

    Figure Lengend Snippet: Western blots depicting the expression of decorin and biglycan after proteoglycan extraction from non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP). For each Western blot, lane (a) is undigested (U) proteoglycan extract from NCD (mongrel) and lane (c) is from CD (beagle) canine NP. Lanes (b) and (d) depict the detection of the specific small leucine-rich proteoglycan (SLRP) indicated above after digestion (D) with chondroitinase ABC and keratinase in NCD canine ( lane b ) and CD canine ( lane d ). a The decorin core protein is visualized appropriately at the 43 kDa molecular weight after digestion ( lane b ), with a clear, single band found from NCD NP extracts. NP extracts from CD canines ( lane d ) reveal fragmentation of decorin with multiple lower molecular weight bands visualized at 30, 25–20, and 17–15 kDa. b The biglycan core protein provides a clear, single band at the 45 kDa molecular weight in the NCD NP extract; however, the CD NP extract reveals multiple fragments of the core protein visualized at 37–35, 28, and 25 kDa

    Article Snippet: Briefly, 500 μg of protein from NCD and CD samples were digested with 1.0 U of chondroitinase ABC (C3667; Sigma-Aldrich) and 0.05 U of keratinase (G6920; Sigma-Aldrich) overnight at 37 °C.

    Techniques: Western Blot, Expressing, Molecular Weight

    SDS-PAGE and immunoblotting of aggrecan with antibodies to different regions. Proteoglycan from guanidine extracts of OA articular cartilage, treated with keratanase and chondroitinase ABC, was analyzed by electrophoresis on polyacrylamide gels, and the fractionated proteoglycan then transferred to nitrocellulose membranes. Aggrecan was visualized by immunoblotting using antibodies recognizing the G1, G2, CS1 or G3 regions. Cartilage samples were obtained adjacent to the lesion (L), midway between the lesion and the joint margin (M), and remote from the lesion (R) from the femoral condyles. Representative samples from patient 1 are shown. Molecular weights of reference proteins are indicated at the left hand side of the blot. Schematic diagrams depicting the core protein structure of aggrecan fragments of different sizes are included at the right side of the blot

    Journal: BMC Musculoskeletal Disorders

    Article Title: Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis

    doi: 10.1186/s12891-016-0944-8

    Figure Lengend Snippet: SDS-PAGE and immunoblotting of aggrecan with antibodies to different regions. Proteoglycan from guanidine extracts of OA articular cartilage, treated with keratanase and chondroitinase ABC, was analyzed by electrophoresis on polyacrylamide gels, and the fractionated proteoglycan then transferred to nitrocellulose membranes. Aggrecan was visualized by immunoblotting using antibodies recognizing the G1, G2, CS1 or G3 regions. Cartilage samples were obtained adjacent to the lesion (L), midway between the lesion and the joint margin (M), and remote from the lesion (R) from the femoral condyles. Representative samples from patient 1 are shown. Molecular weights of reference proteins are indicated at the left hand side of the blot. Schematic diagrams depicting the core protein structure of aggrecan fragments of different sizes are included at the right side of the blot

    Article Snippet: The dry pellet was dissolved in 50 mM sodium acetate, pH 6.0, and digested with 0.2 mU/ml keratanase II (Seikagaku) overnight at 37 °C.

    Techniques: SDS Page, Electrophoresis

    SDS-PAGE and immunoblotting of aggrecan from different individuals. Proteoglycan from guanidine extracts of OA articular cartilage, treated with keratanase and chondroitinase ABC, were analyzed by electrophoresis on polyacrylamide gels, and the fractionated proteoglycan then transferred to nitrocellulose membranes. Aggrecan was visualized by immunoblotting using an antibody recognizing the G1 region. Cartilage samples were obtained midway between the lesion and the joint margin from the femoral condyles and representative samples from eight individuals (M3-M10) are shown. Molecular weights of reference proteins are indicated at the left hand side of the blot. The migration positions of G1 generated by MMP action (G1-MMP) or aggrecanase action (G1-Agg) were determined using anti-neoepitope antibodies specific for their C-terminal peptide sequences (αG1 MMP and αG1 Agg, respectively). αG1MMP shows both terminal neoepitope and some internal sequence recognition

    Journal: BMC Musculoskeletal Disorders

    Article Title: Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis

    doi: 10.1186/s12891-016-0944-8

    Figure Lengend Snippet: SDS-PAGE and immunoblotting of aggrecan from different individuals. Proteoglycan from guanidine extracts of OA articular cartilage, treated with keratanase and chondroitinase ABC, were analyzed by electrophoresis on polyacrylamide gels, and the fractionated proteoglycan then transferred to nitrocellulose membranes. Aggrecan was visualized by immunoblotting using an antibody recognizing the G1 region. Cartilage samples were obtained midway between the lesion and the joint margin from the femoral condyles and representative samples from eight individuals (M3-M10) are shown. Molecular weights of reference proteins are indicated at the left hand side of the blot. The migration positions of G1 generated by MMP action (G1-MMP) or aggrecanase action (G1-Agg) were determined using anti-neoepitope antibodies specific for their C-terminal peptide sequences (αG1 MMP and αG1 Agg, respectively). αG1MMP shows both terminal neoepitope and some internal sequence recognition

    Article Snippet: The dry pellet was dissolved in 50 mM sodium acetate, pH 6.0, and digested with 0.2 mU/ml keratanase II (Seikagaku) overnight at 37 °C.

    Techniques: SDS Page, Electrophoresis, Migration, Generated, Sequencing

    Western analysis demonstrated that keratanase reduces the phosphorylation of an 80 kDa protein. An anti-phosphotyrosine antibody was used to examine whether keratanase altered the phosphorylation of specific proteins in dentate explants. In control explants treated with HBSS, a strong band was present at ∼80 kDa. This band was not detectable in homogenates from cultures treated with keratanases I+II (KIK2).

    Journal: The Journal of Neuroscience

    Article Title: Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration

    doi: 10.1523/JNEUROSCI.3040-03.2004

    Figure Lengend Snippet: Western analysis demonstrated that keratanase reduces the phosphorylation of an 80 kDa protein. An anti-phosphotyrosine antibody was used to examine whether keratanase altered the phosphorylation of specific proteins in dentate explants. In control explants treated with HBSS, a strong band was present at ∼80 kDa. This band was not detectable in homogenates from cultures treated with keratanases I+II (KIK2).

    Article Snippet: Chondroitin ABC lyase, keratanase I, and keratanase II (Seikagaku America, East Falmouth, MA) were dissolved in HBSS to a final concentration of 10 U/ml for ABC lyase and 1 U/ml for keratanases I and II.

    Techniques: Western Blot

    Western analysis demonstrated that chondroitin ABC lyase and keratanase treatments were effective at removing CS- and KS-GAG sugars from proteoglycans in living hippocampal cultures. Hippocampal cultures were treated with either HBSS (H), ABC lyase (A), or a mixture of keratanases I+II (K). First, Western analysis was performed using the 4S antibody, which only recognizes CSPGs after ABC lyase has removed CS-GAGs but does not recognize intact CSPGs. The 4S Western blot showed no staining in H-treated cultures and a strong band in the A-treated cultures ( n = 3). To demonstrate that there were equal amounts of 4S-positive material in the homogenates of H- and A-treated cultures, aliquots of the same homogenates run in lanes 1 and 2 were post-treated with ABC lyase (ABC Post). The 4S-positve band from H-treated cultures was not significantly different from the band in A-treated cultures when the homogenates were post-treated with ABC lyase ( n = 3, last two lanes). To test the effectiveness of keratanase treatment at removing KS-GAGs from phosphacan, Western analysis was performed on homogenates from slice cultures treated with either H or K. Because the 3H1 antibody recognizes KS-GAGs on phosphacan, the absence of a 3H1-positive band in homogenates from K-treated cultures demonstrated that our protocol was effective at removing KS-GAGs from phosphacan. The same blot was then stripped and reprobed for 3F8, which recognizes phosphacan without KS-GAGs. The intensity of the 3F8-positive band from K-treated cultures was not significantly different from those from H-treated cultures ( n = 3), which demonstrates that keratanase treatment did not degrade non-KS forms of phosphacan.

    Journal: The Journal of Neuroscience

    Article Title: Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration

    doi: 10.1523/JNEUROSCI.3040-03.2004

    Figure Lengend Snippet: Western analysis demonstrated that chondroitin ABC lyase and keratanase treatments were effective at removing CS- and KS-GAG sugars from proteoglycans in living hippocampal cultures. Hippocampal cultures were treated with either HBSS (H), ABC lyase (A), or a mixture of keratanases I+II (K). First, Western analysis was performed using the 4S antibody, which only recognizes CSPGs after ABC lyase has removed CS-GAGs but does not recognize intact CSPGs. The 4S Western blot showed no staining in H-treated cultures and a strong band in the A-treated cultures ( n = 3). To demonstrate that there were equal amounts of 4S-positive material in the homogenates of H- and A-treated cultures, aliquots of the same homogenates run in lanes 1 and 2 were post-treated with ABC lyase (ABC Post). The 4S-positve band from H-treated cultures was not significantly different from the band in A-treated cultures when the homogenates were post-treated with ABC lyase ( n = 3, last two lanes). To test the effectiveness of keratanase treatment at removing KS-GAGs from phosphacan, Western analysis was performed on homogenates from slice cultures treated with either H or K. Because the 3H1 antibody recognizes KS-GAGs on phosphacan, the absence of a 3H1-positive band in homogenates from K-treated cultures demonstrated that our protocol was effective at removing KS-GAGs from phosphacan. The same blot was then stripped and reprobed for 3F8, which recognizes phosphacan without KS-GAGs. The intensity of the 3F8-positive band from K-treated cultures was not significantly different from those from H-treated cultures ( n = 3), which demonstrates that keratanase treatment did not degrade non-KS forms of phosphacan.

    Article Snippet: Chondroitin ABC lyase, keratanase I, and keratanase II (Seikagaku America, East Falmouth, MA) were dissolved in HBSS to a final concentration of 10 U/ml for ABC lyase and 1 U/ml for keratanases I and II.

    Techniques: Western Blot, Staining

    Confocal images show that MFs regenerated in a disorganized manner when hippocampal slice cultures were treated with keratanase. Slices were treated with either chondroitin ABC lyase or keratanase II. MFs were stained by injecting Micro Ruby into the granule cell layer of living slices. A series of 0.5-μm-thick optical sections were taken through the slices using a Leica TCS SP2 confocal microscope. Whereas MFs regenerated their normal pattern within the SL in ABC lyase-treated slices (arrows), MFs in keratanase-treated slices wandered laterally into stratum pyramidale and stratum radiatum. MFs in keratanase-treated cultures also appeared to spread more in the vertical plane of the cultures. Scale bar, 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration

    doi: 10.1523/JNEUROSCI.3040-03.2004

    Figure Lengend Snippet: Confocal images show that MFs regenerated in a disorganized manner when hippocampal slice cultures were treated with keratanase. Slices were treated with either chondroitin ABC lyase or keratanase II. MFs were stained by injecting Micro Ruby into the granule cell layer of living slices. A series of 0.5-μm-thick optical sections were taken through the slices using a Leica TCS SP2 confocal microscope. Whereas MFs regenerated their normal pattern within the SL in ABC lyase-treated slices (arrows), MFs in keratanase-treated slices wandered laterally into stratum pyramidale and stratum radiatum. MFs in keratanase-treated cultures also appeared to spread more in the vertical plane of the cultures. Scale bar, 100 μm.

    Article Snippet: Chondroitin ABC lyase, keratanase I, and keratanase II (Seikagaku America, East Falmouth, MA) were dissolved in HBSS to a final concentration of 10 U/ml for ABC lyase and 1 U/ml for keratanases I and II.

    Techniques: Staining, Microscopy

    ADAMTS12 is an evolutionarily conserved chondroitin sulfate/dermatan sulfate proteoglycan. ( A ) Schematic of ADAMTS12 domain structure. TSR, thrombospondin type 1 repeat; CS, chondroitin sulfate. ( B ) Alignment of human and mouse ADAMTS12 mucin modules showing a high degree of conservation (asterisks show identical residues, residue numbers are shown on the right), potential N-glycosylation sites (bold) and Ser-Gly dipeptide motifs (red) with acidic neighbors, constituting putative glycosaminoglycan attachment sites. ( C ) Sequence of the corresponding module from the Ciona intestinalis ortholog of ADAMTS7 and ADAMTS12. Sequence motifs for N-glycosylation (bold) and Ser-Gly/Gly-Ser dipeptides (red) are shown. ( D ) Representative Western blot (anti-FLAG) of conditioned medium from transiently transfected HEK293F cells shows increased ADAMTS12 band intensity (arrows) following digestion with chondroitinase ABC (C’ABC), but not heparanase or keratanase I II. The blot is representative of n = 3. ( E ) Mutation of Ser 1157 and Ser 1159 (boxed residues in panel B ), but not Ser 1157 alone eliminates enhanced signal after C’ABC digestion and reactivity to an anti–chondroitin sulfate stub antibody (MAB2035) strongly suggestive of chondroitin/dermatan sulfate attachment at Ser 1157 and Ser 1159 . EV, empty vector; WT, wild type.

    Journal: JCI Insight

    Article Title: The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate, tendon-specific protective mechanism against heterotopic ossification

    doi: 10.1172/jci.insight.92941

    Figure Lengend Snippet: ADAMTS12 is an evolutionarily conserved chondroitin sulfate/dermatan sulfate proteoglycan. ( A ) Schematic of ADAMTS12 domain structure. TSR, thrombospondin type 1 repeat; CS, chondroitin sulfate. ( B ) Alignment of human and mouse ADAMTS12 mucin modules showing a high degree of conservation (asterisks show identical residues, residue numbers are shown on the right), potential N-glycosylation sites (bold) and Ser-Gly dipeptide motifs (red) with acidic neighbors, constituting putative glycosaminoglycan attachment sites. ( C ) Sequence of the corresponding module from the Ciona intestinalis ortholog of ADAMTS7 and ADAMTS12. Sequence motifs for N-glycosylation (bold) and Ser-Gly/Gly-Ser dipeptides (red) are shown. ( D ) Representative Western blot (anti-FLAG) of conditioned medium from transiently transfected HEK293F cells shows increased ADAMTS12 band intensity (arrows) following digestion with chondroitinase ABC (C’ABC), but not heparanase or keratanase I II. The blot is representative of n = 3. ( E ) Mutation of Ser 1157 and Ser 1159 (boxed residues in panel B ), but not Ser 1157 alone eliminates enhanced signal after C’ABC digestion and reactivity to an anti–chondroitin sulfate stub antibody (MAB2035) strongly suggestive of chondroitin/dermatan sulfate attachment at Ser 1157 and Ser 1159 . EV, empty vector; WT, wild type.

    Article Snippet: Chondroitinase ABC (C3667, Sigma-Aldrich), keratanase I + II (Seikagaku Corporation), or heparinase I + III (H3917, Sigma-Aldrich) was used for deglycosylation as previously described ( ).

    Techniques: Sequencing, Western Blot, Transfection, Mutagenesis, Plasmid Preparation

    Synthesis of osteoadherin by primary bovine osteoblasts and digestion of the immunoprecipitated osteoadherin with keratanase and N -glycosidase F. Osteoblasts were prepared from bovine bone using the method of Robey ( 41 ) and grown to confluency in Ham's F12 medium containing 10% calf serum. The cells were metabolically labeled in the presence of [ 35 S]sulfate for 6 h. Radiolabeled proteins in the culture medium were immunoprecipitated with the osteoadherin antiserum. The precipitates were digested with two different glycosidases. Digested samples, undigested control, and the whole cell medium were electrophoresed on 4–16% gradient polyacrylamide gels and stained with Coomassie blue. Labeled proteins were detected by fluorography. ( A ) Immunoprecipitated osteoadherin in the left lane, immunoprecipitated osteoadherin digested with keratanase in the middle lane, and nonprecipitated whole culture medium in the right lane. ( B ) Immunoprecipitated osteoadherin in the left lane, and immunoprecipitated osteoadherin digested with N -glycosidase F (N-glycanase) in the right lane. Both digestions were performed according to the recommendations of the manufacturers. The positions of molecular mass markers are indicated by arrows.

    Journal: The Journal of Cell Biology

    Article Title: Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone

    doi:

    Figure Lengend Snippet: Synthesis of osteoadherin by primary bovine osteoblasts and digestion of the immunoprecipitated osteoadherin with keratanase and N -glycosidase F. Osteoblasts were prepared from bovine bone using the method of Robey ( 41 ) and grown to confluency in Ham's F12 medium containing 10% calf serum. The cells were metabolically labeled in the presence of [ 35 S]sulfate for 6 h. Radiolabeled proteins in the culture medium were immunoprecipitated with the osteoadherin antiserum. The precipitates were digested with two different glycosidases. Digested samples, undigested control, and the whole cell medium were electrophoresed on 4–16% gradient polyacrylamide gels and stained with Coomassie blue. Labeled proteins were detected by fluorography. ( A ) Immunoprecipitated osteoadherin in the left lane, immunoprecipitated osteoadherin digested with keratanase in the middle lane, and nonprecipitated whole culture medium in the right lane. ( B ) Immunoprecipitated osteoadherin in the left lane, and immunoprecipitated osteoadherin digested with N -glycosidase F (N-glycanase) in the right lane. Both digestions were performed according to the recommendations of the manufacturers. The positions of molecular mass markers are indicated by arrows.

    Article Snippet: Analysis of Carbohydrate Substituents The osteoadherin protein was immunoprecipitated from [35 S]sulfate- labeled cells (as described above) and digested with keratanase (keratan sulfate, 1,4-β-d -galactanohydrolase, EC 3.2.1.103; Boehringer Mannheim Corp. , Indianapolis, IN) or with N -glycosidase F ( Boehringer Mannheim Corp. ) following the recommendations of the manufacturers.

    Techniques: Immunoprecipitation, Metabolic Labelling, Labeling, Staining

    Immunoblot analysis of purified proteoglycan with monoclonal antibodies TG343 and GCTM-2. Purified proteoglycan in its native form (N), or following treatment with chondroitinase ABC (C) or keratanase (K), was electrophoresed on a 7.5% SDS-PAGE gel under reducing conditions, transferred to Immobilon membrane, and probed with antibody TG343 (left) or GCTM-2 (right). The position of the 205-kDa molecular weight marker is indicated by the arrow.

    Journal: Journal of Anatomy

    Article Title: Biochemical properties of a keratan sulphate/chondroitin sulphate proteoglycan expressed in primate pluripotent stem cells *

    doi: 10.1046/j.1469-7580.2002.00034.x

    Figure Lengend Snippet: Immunoblot analysis of purified proteoglycan with monoclonal antibodies TG343 and GCTM-2. Purified proteoglycan in its native form (N), or following treatment with chondroitinase ABC (C) or keratanase (K), was electrophoresed on a 7.5% SDS-PAGE gel under reducing conditions, transferred to Immobilon membrane, and probed with antibody TG343 (left) or GCTM-2 (right). The position of the 205-kDa molecular weight marker is indicated by the arrow.

    Article Snippet: Purified proteoglycan was subjected to keratanase (keratan sulphate 1,4-β- d -galactohydrolase; EC 3.2.1.103; Pseudomonas sp., ICN Biomedicals Ltd) or chondroitinase (chondroitinase ABC lyase; EC 4.2.2.4; Proteus vulgaris , also from ICN) digestion followed by SDS/PAGE and immunoblotting as described previously ( ).

    Techniques: Purification, SDS Page, Molecular Weight, Marker

    Immunoblotting of purified proteoglycan before (A) or after (B) keratanase digestion.

    Journal: Journal of Anatomy

    Article Title: Biochemical properties of a keratan sulphate/chondroitin sulphate proteoglycan expressed in primate pluripotent stem cells *

    doi: 10.1046/j.1469-7580.2002.00034.x

    Figure Lengend Snippet: Immunoblotting of purified proteoglycan before (A) or after (B) keratanase digestion.

    Article Snippet: Purified proteoglycan was subjected to keratanase (keratan sulphate 1,4-β- d -galactohydrolase; EC 3.2.1.103; Pseudomonas sp., ICN Biomedicals Ltd) or chondroitinase (chondroitinase ABC lyase; EC 4.2.2.4; Proteus vulgaris , also from ICN) digestion followed by SDS/PAGE and immunoblotting as described previously ( ).

    Techniques: Purification