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  • 99
    Millipore chondroitinase abc
    Rescue of chondroitin sulfate neuroinhibition by enzymatic digestion. Representative inverted‐fluorescence confocal z‐stack projections stained for neurofilament H (NF‐H) show the growth of neurites from the original dorsal root ganglion (DRG) cell body cluster. DRGs are embedded in a hydrogel containing 2.5 mg/ml methacrylated hyaluronic acid (MAHA) adjacent to a <t>chondroitinase</t> <t>ABC</t> digested hydrogel containing 2.5 mg/ml MAHA (A), an undigested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml methacrylated chondroitin‐4‐sulfate (MACS‐A) (B), or a chondroitinase ABC digested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml MACS‐A (C). Scale bars are 0.5 mm. Neurite lengths and radial distance are measured from the edge of the inner core gel (white line(s); determined by transmitted light microscopy). Quantification of traced neurite lengths (D), radial distance of neurite extension into the inner core gel (E), and the number of neurites/groups of neurites crossing from the outer gel to the inner gel (F) demonstrate that chondroitinase ABC digestion decreases the neuroinhibition of MACS‐A because all three neurite growth measurements are higher in the digested MAHA/MACS‐A inner gels than in the undigested MAHA/MACS‐A inner gels (* p
    Chondroitinase Abc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Seikagaku chondroitinase abc
    Immature astrocytes injected with <t>chondroitinase</t> <t>ABC</t> promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling
    Chondroitinase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 92/100, based on 1394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AMS Biotechnology chondroitinase abc
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of <t>chondroitinase</t> <t>ABC-,</t> but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
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    89
    Seikagaku protease free chondroitinase abc
    Frame A: Zymograms showing <t>chondroitinase</t> <t>ABC</t> and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa
    Protease Free Chondroitinase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 89/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim chondroitinase abc
    Frame A: Zymograms showing <t>chondroitinase</t> <t>ABC</t> and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa
    Chondroitinase Abc, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Seikagaku chondroitin lyase abc
    Frame A: Zymograms showing <t>chondroitinase</t> <t>ABC</t> and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa
    Chondroitin Lyase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Biomedicals chondroitin abc lyase
    Frame A: Zymograms showing <t>chondroitinase</t> <t>ABC</t> and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa
    Chondroitin Abc Lyase, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 89/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant chondroitinase abc
    Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with <t>chondroitinase</t> <t>ABC.</t> Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P
    Chondroitinase Abc, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Associates of Cape Cod Inc chondroitin abc lyase
    Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with <t>chondroitinase</t> <t>ABC.</t> Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P
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    Seikagaku proteinase free chondroitinase abc
    ELISA characterisation of rPlnD1. A : Immunoreactivity of protein core using monoclonal anti-perlecan domain I (A71) and polyclonal anti-perlecan antibody (CCN-1); B : HS GAG characterization using monoclonal antibodies against HS chains (10E4), HS/heparin (2Q546), heparin (A7.10) and HS stub detection (light bars) after digestion with Hep III (3G10); C : CS GAG chain characterization using monoclonal antibodies against CS tetrasaccharide type A-D (CS-56), CS hexasaccharide type C-C-A (LY111), CS hexasaccharide type C-A-D (MO-225), CS (7D4) and CS stubs (1B5 (unsulfated), 2B6 (4S) and 3B3 (6S)) detected after no treatment (dark bars), or <t>chondroitinase</t> <t>ABC</t> digestion (light bars). Data corrected for background absorbance and presented as mean ± standard deviation (n=3).
    Proteinase Free Chondroitinase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Seikagaku chondroitinase abc chabc
    ELISA characterisation of rPlnD1. A : Immunoreactivity of protein core using monoclonal anti-perlecan domain I (A71) and polyclonal anti-perlecan antibody (CCN-1); B : HS GAG characterization using monoclonal antibodies against HS chains (10E4), HS/heparin (2Q546), heparin (A7.10) and HS stub detection (light bars) after digestion with Hep III (3G10); C : CS GAG chain characterization using monoclonal antibodies against CS tetrasaccharide type A-D (CS-56), CS hexasaccharide type C-C-A (LY111), CS hexasaccharide type C-A-D (MO-225), CS (7D4) and CS stubs (1B5 (unsulfated), 2B6 (4S) and 3B3 (6S)) detected after no treatment (dark bars), or <t>chondroitinase</t> <t>ABC</t> digestion (light bars). Data corrected for background absorbance and presented as mean ± standard deviation (n=3).
    Chondroitinase Abc Chabc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Acorda antibody against chondroitinase abc
    Expression of <t>chondroitinase</t> in rat brain from different LVs at different times: coronal sections stained with Ab-2B6 as in Fig. 3 . Scale bar, 1 mm. (a) LV-P, 2 weeks: there is widespread digestion on both sides (red arrows). White arrow marks the approximate position of the injection. (b) LV-D, 4 weeks: more circumscribed digestion within ∼0.5 mm of the injection track, but also strong digestion within the corpus callosum extending to the contralateral side. (c) LV-C, 8 weeks: patchy digestion in various regions, especially ipsilateral hippocampus (Hipp) and thalamus (Thal). (d) PBS-injected control, 4 weeks: no specific immunoreactivity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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    Seikagaku chondroitinase csase abc
    Expression of <t>chondroitinase</t> in rat brain from different LVs at different times: coronal sections stained with Ab-2B6 as in Fig. 3 . Scale bar, 1 mm. (a) LV-P, 2 weeks: there is widespread digestion on both sides (red arrows). White arrow marks the approximate position of the injection. (b) LV-D, 4 weeks: more circumscribed digestion within ∼0.5 mm of the injection track, but also strong digestion within the corpus callosum extending to the contralateral side. (c) LV-C, 8 weeks: patchy digestion in various regions, especially ipsilateral hippocampus (Hipp) and thalamus (Thal). (d) PBS-injected control, 4 weeks: no specific immunoreactivity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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    Image Search Results


    Rescue of chondroitin sulfate neuroinhibition by enzymatic digestion. Representative inverted‐fluorescence confocal z‐stack projections stained for neurofilament H (NF‐H) show the growth of neurites from the original dorsal root ganglion (DRG) cell body cluster. DRGs are embedded in a hydrogel containing 2.5 mg/ml methacrylated hyaluronic acid (MAHA) adjacent to a chondroitinase ABC digested hydrogel containing 2.5 mg/ml MAHA (A), an undigested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml methacrylated chondroitin‐4‐sulfate (MACS‐A) (B), or a chondroitinase ABC digested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml MACS‐A (C). Scale bars are 0.5 mm. Neurite lengths and radial distance are measured from the edge of the inner core gel (white line(s); determined by transmitted light microscopy). Quantification of traced neurite lengths (D), radial distance of neurite extension into the inner core gel (E), and the number of neurites/groups of neurites crossing from the outer gel to the inner gel (F) demonstrate that chondroitinase ABC digestion decreases the neuroinhibition of MACS‐A because all three neurite growth measurements are higher in the digested MAHA/MACS‐A inner gels than in the undigested MAHA/MACS‐A inner gels (* p

    Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

    Article Title: Development of an In Vitro Intervertebral Disc Innervation Model to Screen Neuroinhibitory Biomaterials

    doi: 10.1002/jor.24557

    Figure Lengend Snippet: Rescue of chondroitin sulfate neuroinhibition by enzymatic digestion. Representative inverted‐fluorescence confocal z‐stack projections stained for neurofilament H (NF‐H) show the growth of neurites from the original dorsal root ganglion (DRG) cell body cluster. DRGs are embedded in a hydrogel containing 2.5 mg/ml methacrylated hyaluronic acid (MAHA) adjacent to a chondroitinase ABC digested hydrogel containing 2.5 mg/ml MAHA (A), an undigested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml methacrylated chondroitin‐4‐sulfate (MACS‐A) (B), or a chondroitinase ABC digested hydrogel containing 2.25 mg/ml MAHA plus 10 mg/ml MACS‐A (C). Scale bars are 0.5 mm. Neurite lengths and radial distance are measured from the edge of the inner core gel (white line(s); determined by transmitted light microscopy). Quantification of traced neurite lengths (D), radial distance of neurite extension into the inner core gel (E), and the number of neurites/groups of neurites crossing from the outer gel to the inner gel (F) demonstrate that chondroitinase ABC digestion decreases the neuroinhibition of MACS‐A because all three neurite growth measurements are higher in the digested MAHA/MACS‐A inner gels than in the undigested MAHA/MACS‐A inner gels (* p

    Article Snippet: Gels were washed with HBSS (BW10–543F; Thermo Fisher Scientific), incubated with 2.5U chondroitinase ABC (C3667; MilliporeSigma) in 2 ml HBSS plus 61 mM sodium acetate (W302406; MilliporeSigma) for 3 h at 37°C, and washed 9× 15 min with PBS before being stored in PBS at 4°C overnight.

    Techniques: Fluorescence, Staining, Magnetic Cell Separation, Light Microscopy

    Immature astrocytes injected with chondroitinase ABC promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Immature astrocytes injected with chondroitinase ABC promote regeneration of p75+ axons. Immature astrocytes were infected with a GFP lentivirus prior to transplantation into the cingulum microlesion and were therefore identified by GFP immunolabeling

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Injection, Infection, Transplantation Assay, Immunolabeling

    Quantification of axonal regeneration demonstrates that immature astrocytes combined with chondroitinase ABC would be the best treatment for brain injuries. Axonal regeneration was quantified by counting the number of axons crossing the proximal edge

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Quantification of axonal regeneration demonstrates that immature astrocytes combined with chondroitinase ABC would be the best treatment for brain injuries. Axonal regeneration was quantified by counting the number of axons crossing the proximal edge

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques:

    Pre-treatment of gradient spots with chondroitinase ABC enhances axon rim crossing of DRG neurons cultured on immature astrocytes. Adult DRG neurons were cultured for 2 days on immature (A–C) or mature astrocytes (D–F) which had been plated

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Pre-treatment of gradient spots with chondroitinase ABC enhances axon rim crossing of DRG neurons cultured on immature astrocytes. Adult DRG neurons were cultured for 2 days on immature (A–C) or mature astrocytes (D–F) which had been plated

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Cell Culture

    The p75+ axons in chondroitinase ABC treated microlesions are not capable of regenerating. Microlesions were performed in the cingulum and treated with a single injection of 5U/ml chondroitinase ABC. Chondroitinase treatment resulted in 2B6 immunolabelling

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: The p75+ axons in chondroitinase ABC treated microlesions are not capable of regenerating. Microlesions were performed in the cingulum and treated with a single injection of 5U/ml chondroitinase ABC. Chondroitinase treatment resulted in 2B6 immunolabelling

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Injection

    Immature astrocytes are capable of growing on high levels of CSPGs. Immature and mature astrocytes were cultured on gradient spots of a mixture of CSPG and laminin for 1 day, 3 days and 5 days (A), or on spots pre-treated with chondroitinase ABC for 5

    Journal: Developmental neurobiology

    Article Title: Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    doi: 10.1002/dneu.20820

    Figure Lengend Snippet: Immature astrocytes are capable of growing on high levels of CSPGs. Immature and mature astrocytes were cultured on gradient spots of a mixture of CSPG and laminin for 1 day, 3 days and 5 days (A), or on spots pre-treated with chondroitinase ABC for 5

    Article Snippet: Animal groups were defined as: control (animals that received an injection of saline), chondroitinase (animals that received an injection of chondroitinase ABC), immature astrocytes (animals that received an injection of immature astrocytes), immature astrocytes and chondroitinase 5 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 5 days later), immature astrocytes and chondroitinase 7 days (animals that received an injection of immature astrocytes resuspended in a solution of chondroitinase ABC, and were perfused 7 days later).

    Techniques: Cell Culture

    CPG-1 through -6 behave as CSPGs in COS-7 cells. CPG-1 through -6 were expressed as Myc-tagged recombinant proteins in COS-7 cells. Proteoglycans from conditioned media were purified by anion-exchange chromatography (see Materials and Methods). Samples were digested with chondroitinase ABC (+) or left untreated (−), separated by SDS-PAGE, and Western blotted with an anti-Myc mAb.

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: CPG-1 through -6 behave as CSPGs in COS-7 cells. CPG-1 through -6 were expressed as Myc-tagged recombinant proteins in COS-7 cells. Proteoglycans from conditioned media were purified by anion-exchange chromatography (see Materials and Methods). Samples were digested with chondroitinase ABC (+) or left untreated (−), separated by SDS-PAGE, and Western blotted with an anti-Myc mAb.

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: Recombinant, Purification, Chromatography, SDS Page, Western Blot

    C. elegans CPGs identified by BEMAD/MUDPIT. CPGs were purified from worm extracts and identified by mass spectrometry analysis. Nine independent purifications resulted in identification of nine CPGs, most of which were identified in multiple runs. The predicted masses are based on amino acid sequence, and the apparent masses are based on SDS-PAGE migration of recombinant proteins expressed in COS-7 cells after digestion with chondroitinase ABC and reduction. Putative glycosylation sites (short vertical lines) consist of Ser-Gly dipeptides flanked by one or more acidic amino acids. Identified sites (long vertical lines) represent serine residues modified with DTT by the BEMAD method (see Materials and methods). Genes enriched for germline expression were identified by Reinke et al. (2000) . Schematic drawings of each CPG are shown. Gray circles indicate signal peptides, black ovals are peritrophin-A chitin binding domains, diagonally hatched boxes identify C-type lectin domains, and dotted lines below the protein indicate peptide coverage discovered by mass spectrometry.

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: C. elegans CPGs identified by BEMAD/MUDPIT. CPGs were purified from worm extracts and identified by mass spectrometry analysis. Nine independent purifications resulted in identification of nine CPGs, most of which were identified in multiple runs. The predicted masses are based on amino acid sequence, and the apparent masses are based on SDS-PAGE migration of recombinant proteins expressed in COS-7 cells after digestion with chondroitinase ABC and reduction. Putative glycosylation sites (short vertical lines) consist of Ser-Gly dipeptides flanked by one or more acidic amino acids. Identified sites (long vertical lines) represent serine residues modified with DTT by the BEMAD method (see Materials and methods). Genes enriched for germline expression were identified by Reinke et al. (2000) . Schematic drawings of each CPG are shown. Gray circles indicate signal peptides, black ovals are peritrophin-A chitin binding domains, diagonally hatched boxes identify C-type lectin domains, and dotted lines below the protein indicate peptide coverage discovered by mass spectrometry.

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: Purification, Mass Spectrometry, Sequencing, SDS Page, Migration, Recombinant, Modification, Expressing, Binding Assay

    C. elegans express multiple CPGs. (A) Glycopeptides were treated with alkali to produce free glycans, which were then analyzed by gel filtration HPLC (see Materials and methods). Untreated sample eluted as two peaks (indicated by squares), whereas after chondroitinase ABC all the material eluted as disaccharides (indicated by circles). V i , elution position of [1- 3 H]glucose; V o , elution position of blue dextran. Shark cartilage chondroitin sulfate (∼20 kD) was run as a standard. (B) Crude extracts of adult worms (lanes 1–3) or embryos (lane 4) were digested with chondroitinase ABC (lanes 2–4) and separated by SDS-PAGE. Western blotting was performed with mAb 1-B-5, which recognizes a neoepitope generated by chondroitinase digestion. The undigested control (lane 1) demonstrates the requirement of the antibody for chondroitin stubs. Lanes 2 and 4, 10-μg protein; lanes 1 and 3, 40-μg protein. The carats indicate the position of protein bands that arose only after chondroitinase ABC digestion.

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: C. elegans express multiple CPGs. (A) Glycopeptides were treated with alkali to produce free glycans, which were then analyzed by gel filtration HPLC (see Materials and methods). Untreated sample eluted as two peaks (indicated by squares), whereas after chondroitinase ABC all the material eluted as disaccharides (indicated by circles). V i , elution position of [1- 3 H]glucose; V o , elution position of blue dextran. Shark cartilage chondroitin sulfate (∼20 kD) was run as a standard. (B) Crude extracts of adult worms (lanes 1–3) or embryos (lane 4) were digested with chondroitinase ABC (lanes 2–4) and separated by SDS-PAGE. Western blotting was performed with mAb 1-B-5, which recognizes a neoepitope generated by chondroitinase digestion. The undigested control (lane 1) demonstrates the requirement of the antibody for chondroitin stubs. Lanes 2 and 4, 10-μg protein; lanes 1 and 3, 40-μg protein. The carats indicate the position of protein bands that arose only after chondroitinase ABC digestion.

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: Filtration, High Performance Liquid Chromatography, SDS Page, Western Blot, Generated

    CPG-1 and -2 bind chitin and exist as CPGs in vivo. (A) Proteoglycan extracts of vector and RNAi-treated animals were digested with chondroitinase ABC, analyzed by SDS-PAGE, and Western blotted with the 1B5 mAb that recognizes the chondroitin stub remaining after enzyme digestion. cpg-2(RNAi) reduced the bands at 120, ∼80, and ∼45 kD (arrowheads). (B) 20 μg of total worm extract (input) was incubated with chitin beads. Four bands specifically bound chitin (arrowheads), but the 60-kD band did not. (C) Proteoglycan extracts from RNAi-treated animals were enriched by affinity chromatography on chitin beads. cpg-1(RNAi) reduced the band at ∼150 kD (open arrowhead), whereas cpg-2(RNAi) reduced the bands at 120, ∼80, and 45 kD (filled arrowheads).

    Journal: The Journal of Cell Biology

    Article Title: Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2

    doi: 10.1083/jcb.200603003

    Figure Lengend Snippet: CPG-1 and -2 bind chitin and exist as CPGs in vivo. (A) Proteoglycan extracts of vector and RNAi-treated animals were digested with chondroitinase ABC, analyzed by SDS-PAGE, and Western blotted with the 1B5 mAb that recognizes the chondroitin stub remaining after enzyme digestion. cpg-2(RNAi) reduced the bands at 120, ∼80, and ∼45 kD (arrowheads). (B) 20 μg of total worm extract (input) was incubated with chitin beads. Four bands specifically bound chitin (arrowheads), but the 60-kD band did not. (C) Proteoglycan extracts from RNAi-treated animals were enriched by affinity chromatography on chitin beads. cpg-1(RNAi) reduced the band at ∼150 kD (open arrowhead), whereas cpg-2(RNAi) reduced the bands at 120, ∼80, and 45 kD (filled arrowheads).

    Article Snippet: Western blotting Proteoglycan samples (10 μL) were digested with 10 mU chondroitinase ABC (Seikagaku) and/or 2.5 mU heparin lyase II (Sigma-Aldrich) for 3–5 h at 37°C.

    Techniques: In Vivo, Plasmid Preparation, SDS Page, Western Blot, Incubation, Affinity Chromatography

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

    Journal: Cell Research

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

    doi: 10.1038/cr.2015.29

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

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

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

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

    Journal: Cell Research

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

    doi: 10.1038/cr.2015.29

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

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

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

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

    Journal: Cell Research

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

    doi: 10.1038/cr.2015.29

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

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

    Techniques: Western Blot, Activity Assay

    The effect of heparanase on exosome production depends on the modification of the heparan sulfate (HS) on syndecans. (A) The role of heparanase enzymatic activity on exosome production was investigated by comparing MCF-7 cells stably expressing wild-type heparanase (WT), catalytically dead heparanase (Cat) or empty vector (Φ) in western blot, in the absence (−) or presence (+) of 10 nM exogenously added proheparanase. (B) The importance of HS was analyzed by treating MCF-7 cells with RNAi targeting EXT1 and EXT2 (KD). Non-targeting RNAi (NT) was used as a control. Cells were challenged with 10 nM proheparanase (+) or left untreated (−). Heparanase activity, reducing the HS on syndecan, was apparent from the migration of chondroitinase ABC-treated syndecan-1 present in cell lysates (SDC1 with HS). EXT1 and EXT2 knockdown leads to the appearance of syndecan-1 that is not substituted with HS (a band running slightly > 70 kDa, after chondroitinase ABC digestion only), not detectable in cells treated with non-targeting RNAi, where all syndecan is substituted with HS (and is larger than SDC FL). A small amount of the syndecan-1 still carried HS and was affected by heparanase addition (yielding a band slightly > 100 kDa, after chondroitinase ABC digestion only), indicating an incomplete knockdown of EXT1 and EXT2. Western blots are representative of three independent experiments. (C) Quantification of the effect of EXT1 and EXT2 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF, CD63 and flotillin-1 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi and heparanase, dark gray bars; EXT1 and EXT2 RNAi, light gray bars; EXT1 and EXT2 RNAi and heparanase, white bars). Values are relative to the exosomal levels measured in cells treated with non-targeting RNAi and in the absence of exogenously added proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

    Journal: Cell Research

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

    doi: 10.1038/cr.2015.29

    Figure Lengend Snippet: The effect of heparanase on exosome production depends on the modification of the heparan sulfate (HS) on syndecans. (A) The role of heparanase enzymatic activity on exosome production was investigated by comparing MCF-7 cells stably expressing wild-type heparanase (WT), catalytically dead heparanase (Cat) or empty vector (Φ) in western blot, in the absence (−) or presence (+) of 10 nM exogenously added proheparanase. (B) The importance of HS was analyzed by treating MCF-7 cells with RNAi targeting EXT1 and EXT2 (KD). Non-targeting RNAi (NT) was used as a control. Cells were challenged with 10 nM proheparanase (+) or left untreated (−). Heparanase activity, reducing the HS on syndecan, was apparent from the migration of chondroitinase ABC-treated syndecan-1 present in cell lysates (SDC1 with HS). EXT1 and EXT2 knockdown leads to the appearance of syndecan-1 that is not substituted with HS (a band running slightly > 70 kDa, after chondroitinase ABC digestion only), not detectable in cells treated with non-targeting RNAi, where all syndecan is substituted with HS (and is larger than SDC FL). A small amount of the syndecan-1 still carried HS and was affected by heparanase addition (yielding a band slightly > 100 kDa, after chondroitinase ABC digestion only), indicating an incomplete knockdown of EXT1 and EXT2. Western blots are representative of three independent experiments. (C) Quantification of the effect of EXT1 and EXT2 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF, CD63 and flotillin-1 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi and heparanase, dark gray bars; EXT1 and EXT2 RNAi, light gray bars; EXT1 and EXT2 RNAi and heparanase, white bars). Values are relative to the exosomal levels measured in cells treated with non-targeting RNAi and in the absence of exogenously added proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

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

    Techniques: Modification, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Migration

    Frame A: Zymograms showing chondroitinase ABC and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: Frame A: Zymograms showing chondroitinase ABC and testicular hyaluronidase digestions of the 230 and 72 kDa gelatinases. After digestion with either enzyme, 230 kDa gelatinase band showed a mobility shift, whereas no shift was observed for the 72 kDa

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: Mobility Shift

    MMP enzyme assay using peptide substrate. The effects of APMA activation and chondroitinase ABC treatment on the enzyme activity of the 230 kDa gelatinase were tested using a quenched fluorescent synthetic peptide substrate. APMA activated the latent

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: MMP enzyme assay using peptide substrate. The effects of APMA activation and chondroitinase ABC treatment on the enzyme activity of the 230 kDa gelatinase were tested using a quenched fluorescent synthetic peptide substrate. APMA activated the latent

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: Enzymatic Assay, Activation Assay, Activity Assay

    Macromolecular composition of the 230 kDa gelatinase. Frame A shows a chondroitinase ABC digestion of the 230 kDa gelatinase on SDS-PAGE stained with Coomassie colloidal blue stain. Lanes M, molecular weight standards; 1, chondroitinase ABC enzyme alone;

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: Macromolecular composition of the 230 kDa gelatinase. Frame A shows a chondroitinase ABC digestion of the 230 kDa gelatinase on SDS-PAGE stained with Coomassie colloidal blue stain. Lanes M, molecular weight standards; 1, chondroitinase ABC enzyme alone;

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: SDS Page, Staining, Molecular Weight

    Influence of TIMPs and chondroitinase ABC treatment on 230 kDa gelatinase activity. In all assays the 230 kDa gelatinase was APMA activated and incubated with the peptide substrate in presence of 500 ng of TIMP-1 or TIMP-2 either without (frame A) or

    Journal: Archives of biochemistry and biophysics

    Article Title: Matrix metalloproteinase-9 in a unique proteoglycan form in avian embryonic growth plate cartilage

    doi: 10.1016/j.abb.2012.02.003

    Figure Lengend Snippet: Influence of TIMPs and chondroitinase ABC treatment on 230 kDa gelatinase activity. In all assays the 230 kDa gelatinase was APMA activated and incubated with the peptide substrate in presence of 500 ng of TIMP-1 or TIMP-2 either without (frame A) or

    Article Snippet: 230 kDa enzyme was digested with protease-free chondroitinase ABC (40 μU) (Seikagaku Co., Tokyo, Japan) in chondroitinase buffer containing 50 mM Tris, pH 8.0, 30 mM sodium acetate, 0.02% (v/v) Brij-35, 0.02% (w/v) NaN3 and 0.05% (w/v) protease free BSA at 37°C for 2 h. Digested proteins were separated by SDS-PAGE and detected by Coomassie colloidal blue stain (OWL Separation Systems, Woburn, MA).

    Techniques: Activity Assay, Incubation

    GPC1 expression in tumor xenografts. ( A ) Immunoblotting. Tumors from sham-transfected and GAS PANC-1 cells (GAS6 and GAS7) were homogenized and incubated with control buffer, heparitinase, or heparitinase and chondroitinase ABC (Ch-ABC). Protein lysates were subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. ( B ) Densitometric analysis. Frozen lysates for immunoblotting from 6 sham, 5 GAS6, and 4 GAS7 tumors were subjected to densitometry. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

    doi: 10.1172/JCI32412

    Figure Lengend Snippet: GPC1 expression in tumor xenografts. ( A ) Immunoblotting. Tumors from sham-transfected and GAS PANC-1 cells (GAS6 and GAS7) were homogenized and incubated with control buffer, heparitinase, or heparitinase and chondroitinase ABC (Ch-ABC). Protein lysates were subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. ( B ) Densitometric analysis. Frozen lysates for immunoblotting from 6 sham, 5 GAS6, and 4 GAS7 tumors were subjected to densitometry. * P

    Article Snippet: The following materials were purchased from the following companies: DMEM, RPMI 1640, and trypsin-EDTA from Mediatech Inc.; FBS from Omega Scientific Inc.; G418 from GIBCO Laboratories; Noble agar from Difco Laboratories; recombinant human EGF from Chemicon; Heparitinase and Chondroitinase ABC Protease Free from Seikagaku Corporation; One Solution reagent and anti–phospho-MAPK antibody from Promega; Immobilon-P PVDF membranes from Millipore Corp.; RNeasy Mini Kit from Qiagen; phosphatidylinositol-specific phospholipase C (PI-PLC) from Molecular Probes; magnetic dynabeads from Dynal Biotech from Invitrogen; SulfoLink Coupling Gel from Pierce Biotechnology; anti–ERK-2 antibody, anti–angiopoietin-1 and -2 antibodies, and anti-Tie2 antibody from Santa Cruz Biotechnology Inc.; anti-CD31 rat anti-mouse monoclonal antibody (catalog no. 553370) and anti-VE cadherin antibody from Pharmingen; anti-PCNA monoclonal antibody from Novocastra; Antigen Unmasking Solution from Vector Laboratories; ApopTag in situ apoptosis detection kit from Chemicon; oligonucleotide primers from Applied Biosystems; PANC-1 human pancreatic cancer cells and COS-7 cells from American Type Culture Collection.

    Techniques: Expressing, Transfection, Incubation, Affinity Purification

    Effects of GPC1 antisense expression on GPC1 protein levels. ( A ) Effects of enzymatic treatment on immunoblotting. Sham-transfected cells and GPC1 antisense–expressing cells (clone GAS6) were incubated in the absence or presence of the indicated enzymes and subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. ( B ) Total cell lysates from sham-transfected and from both antisense-expressing clones as well as the corresponding conditioned media were incubated with heparitinase and chondroitinase ABC and subjected to immunoblotting as described above. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. Each panel is representative of 2 distinct experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells

    doi: 10.1172/JCI32412

    Figure Lengend Snippet: Effects of GPC1 antisense expression on GPC1 protein levels. ( A ) Effects of enzymatic treatment on immunoblotting. Sham-transfected cells and GPC1 antisense–expressing cells (clone GAS6) were incubated in the absence or presence of the indicated enzymes and subjected to immunoblotting using affinity-purified rabbit anti-GPC1 antibody as reported in Methods. ( B ) Total cell lysates from sham-transfected and from both antisense-expressing clones as well as the corresponding conditioned media were incubated with heparitinase and chondroitinase ABC and subjected to immunoblotting as described above. Membranes from cell lysates were reprobed with an anti-ERK2 antibody to confirm equivalent loading of lanes. Each panel is representative of 2 distinct experiments.

    Article Snippet: The following materials were purchased from the following companies: DMEM, RPMI 1640, and trypsin-EDTA from Mediatech Inc.; FBS from Omega Scientific Inc.; G418 from GIBCO Laboratories; Noble agar from Difco Laboratories; recombinant human EGF from Chemicon; Heparitinase and Chondroitinase ABC Protease Free from Seikagaku Corporation; One Solution reagent and anti–phospho-MAPK antibody from Promega; Immobilon-P PVDF membranes from Millipore Corp.; RNeasy Mini Kit from Qiagen; phosphatidylinositol-specific phospholipase C (PI-PLC) from Molecular Probes; magnetic dynabeads from Dynal Biotech from Invitrogen; SulfoLink Coupling Gel from Pierce Biotechnology; anti–ERK-2 antibody, anti–angiopoietin-1 and -2 antibodies, and anti-Tie2 antibody from Santa Cruz Biotechnology Inc.; anti-CD31 rat anti-mouse monoclonal antibody (catalog no. 553370) and anti-VE cadherin antibody from Pharmingen; anti-PCNA monoclonal antibody from Novocastra; Antigen Unmasking Solution from Vector Laboratories; ApopTag in situ apoptosis detection kit from Chemicon; oligonucleotide primers from Applied Biosystems; PANC-1 human pancreatic cancer cells and COS-7 cells from American Type Culture Collection.

    Techniques: Expressing, Transfection, Incubation, Affinity Purification, Clone Assay

    Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with chondroitinase ABC. Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P

    Journal: The Journal of Neuroscience

    Article Title: NG2 Glial Cells Provide a Favorable Substrate for Growing Axons

    doi: 10.1523/JNEUROSCI.4247-05.2006

    Figure Lengend Snippet: Effects of increased NG2 expression on neurite growth. A–D , Comparison of the level of NG2 expression in control NG2 cells and in NG2 cells infected with adeno-NG2. A , B , Surface immunofluorescence labeling (live-cell labeling) for NG2 on uninfected ( A ) and adeno-NG2-infected cells ( B ; moi 5). Images were acquired under identical exposure conditions below pixel saturation. Scale bar: (in B ) A , B , 12 μm. C , Quantification of fluorescence intensity of uninfected NG2 cells and NG2 cells infected with adeno-NG2. Cells transduced with adeno-NG2 expressed fivefold more surface NG2 than uninfected NG2 cells. D , Western blots comparing the amount of NG2 in extracts of uninfected and adeno-NG2-infected NG2 cells. The same blot was probed with antibodies to NG2 (top) and GAPDH (bottom) chABC+: extracts treated with chondroitinase ABC. Smaller amounts of protein extracts from adeno-NG2-infected cells were loaded to allow for densitometric analyses of the samples within the dynamic ranges of the pixels, as seen by the weaker GAPDH bands in lanes 3 and 4. The positions of molecular weight standards are indicated on the left. E–I , Response of P1 hippocampal neurons to NG2 cells expressing elevated levels of NG2. E , F , Double-immunofluorescence labeling for NG2 (red) and βIII tubulin (green) to illustrate the spatial relationship between neurites and NG2 cells expressing normal ( E ) or increased levels of surface NG2 after adeno-NG2 transduction ( F ; moi 5). Scale bar: (in F ) E , F , 24 μm. G , Percentage of neurite tips contacting uninfected or adeno-NG2-infected NG2 cells. ns, No statistically significant difference between the number of neurite tips contacting uninfected and adeno-NG2-infected NG2 cells ( P = 0.239, t test). H , Total neurite lengths of neurons grown on uninfected or adeno-NG2-infected NG2 cells. ns, No significant difference ( P > 0.05, one-way ANOVA). They were significantly longer than neurite lengths on PLL ( P

    Article Snippet: Some extracts were treated with 0.125 U of chondroitinase ABC (MP Biomedicals, Aurora, OH) at room temperature for an additional 15 min. Extracts were then centrifuged at 15,000 × g for 5 min at 4°C to remove insoluble material, and supernatants were heated at 80°C for 10 min in reducing sample buffer (6% SDS, 40% glycerol, and 0.2 mg/ml BPB, and 100 m m DTT in 125 m m Tris-HCl, pH 6.8).

    Techniques: Expressing, Infection, Immunofluorescence, Labeling, Fluorescence, Transduction, Western Blot, Molecular Weight

    ELISA characterisation of rPlnD1. A : Immunoreactivity of protein core using monoclonal anti-perlecan domain I (A71) and polyclonal anti-perlecan antibody (CCN-1); B : HS GAG characterization using monoclonal antibodies against HS chains (10E4), HS/heparin (2Q546), heparin (A7.10) and HS stub detection (light bars) after digestion with Hep III (3G10); C : CS GAG chain characterization using monoclonal antibodies against CS tetrasaccharide type A-D (CS-56), CS hexasaccharide type C-C-A (LY111), CS hexasaccharide type C-A-D (MO-225), CS (7D4) and CS stubs (1B5 (unsulfated), 2B6 (4S) and 3B3 (6S)) detected after no treatment (dark bars), or chondroitinase ABC digestion (light bars). Data corrected for background absorbance and presented as mean ± standard deviation (n=3).

    Journal: BMC Biotechnology

    Article Title: Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

    doi: 10.1186/1472-6750-12-60

    Figure Lengend Snippet: ELISA characterisation of rPlnD1. A : Immunoreactivity of protein core using monoclonal anti-perlecan domain I (A71) and polyclonal anti-perlecan antibody (CCN-1); B : HS GAG characterization using monoclonal antibodies against HS chains (10E4), HS/heparin (2Q546), heparin (A7.10) and HS stub detection (light bars) after digestion with Hep III (3G10); C : CS GAG chain characterization using monoclonal antibodies against CS tetrasaccharide type A-D (CS-56), CS hexasaccharide type C-C-A (LY111), CS hexasaccharide type C-A-D (MO-225), CS (7D4) and CS stubs (1B5 (unsulfated), 2B6 (4S) and 3B3 (6S)) detected after no treatment (dark bars), or chondroitinase ABC digestion (light bars). Data corrected for background absorbance and presented as mean ± standard deviation (n=3).

    Article Snippet: Endoglycosidase enzymes, proteinase free chondroitinase ABC (C’ase ABC) and heparinase III (Hep III) (EC 4.2.2.8), were purchased from Seikagaku Corp., Tokyo, Japan.

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Expression of chondroitinase in rat brain from different LVs at different times: coronal sections stained with Ab-2B6 as in Fig. 3 . Scale bar, 1 mm. (a) LV-P, 2 weeks: there is widespread digestion on both sides (red arrows). White arrow marks the approximate position of the injection. (b) LV-D, 4 weeks: more circumscribed digestion within ∼0.5 mm of the injection track, but also strong digestion within the corpus callosum extending to the contralateral side. (c) LV-C, 8 weeks: patchy digestion in various regions, especially ipsilateral hippocampus (Hipp) and thalamus (Thal). (d) PBS-injected control, 4 weeks: no specific immunoreactivity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Journal of Neuroscience Methods

    Article Title: Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

    doi: 10.1016/j.jneumeth.2011.08.003

    Figure Lengend Snippet: Expression of chondroitinase in rat brain from different LVs at different times: coronal sections stained with Ab-2B6 as in Fig. 3 . Scale bar, 1 mm. (a) LV-P, 2 weeks: there is widespread digestion on both sides (red arrows). White arrow marks the approximate position of the injection. (b) LV-D, 4 weeks: more circumscribed digestion within ∼0.5 mm of the injection track, but also strong digestion within the corpus callosum extending to the contralateral side. (c) LV-C, 8 weeks: patchy digestion in various regions, especially ipsilateral hippocampus (Hipp) and thalamus (Thal). (d) PBS-injected control, 4 weeks: no specific immunoreactivity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: The antibody against chondroitinase ABC was kindly given by Acorda Inc.

    Techniques: Expressing, Staining, Injection

    Immunocytochemistry showing expression of the vectors in tissue culture cells. Cells were transduced with lentiviral vectors, fixed, reacted with antibody against chondroitinase, and visualized with peroxidase diaminobenzidine reaction. Scale bars, 100 μm. Top row: LV-D in HEK293T cells, Neu7 cells, and SCTM41 cells. Middle rowLV-P in HEK293T cells, Neu7 cells, and primary astrocytes. Bottom row: negative control cultures with no vector.

    Journal: Journal of Neuroscience Methods

    Article Title: Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

    doi: 10.1016/j.jneumeth.2011.08.003

    Figure Lengend Snippet: Immunocytochemistry showing expression of the vectors in tissue culture cells. Cells were transduced with lentiviral vectors, fixed, reacted with antibody against chondroitinase, and visualized with peroxidase diaminobenzidine reaction. Scale bars, 100 μm. Top row: LV-D in HEK293T cells, Neu7 cells, and SCTM41 cells. Middle rowLV-P in HEK293T cells, Neu7 cells, and primary astrocytes. Bottom row: negative control cultures with no vector.

    Article Snippet: The antibody against chondroitinase ABC was kindly given by Acorda Inc.

    Techniques: Immunocytochemistry, Expressing, Transduction, Negative Control, Plasmid Preparation

    In vivo LV-ChABC injection into the rat cortex results in chondroitinase activity and the activity is also transported along axons. Injection was into deep layers of cortex on the left. Scale bar, 1 mm. (a–c) LV-C, 2 weeks: adjacent coronal sections from one brain stained for chondroitinase-produced stub (a), perineuronal net (b) and chondroitin sulfate (c). Chondroitinase digestion (area outlined with red arrows) is seen around the injection site and in the cortical white matter and adjacent hippocampus. On the original of (a), a faint column of reactivity can also be seen in the contralateral cortex. M, midline. (d–f) LV-C, 4 weeks: a similar series of sections, showing that chondroitinase digestion has extended along axon tracts and to the contralateral cortex. (g,h) LV-GFP, 4 weeks: similar sections from a brain injected with a lentivector encoding farnesyl-GFP; 4 weeks; stained with antibody against GFP. (g) Cortex near injection site, showing strong expression in astrocytes at surface (A), and oligodendrocytes in white matter/corpus callosum (O), and large numbers of neuronal fibers (N). (h) High-power views of contralateral side showing GFP-positive axons that have crossed in the corpus callosum and (inset) ascending in the cortex opposite the injection site. (i) Enzyme activity assay of functional chondroitinase level 4 weeks after LV injection into the cortex. Disks were loaded with equal amounts of chondroitin sulfate (CS) and each was incubated with extract of cortex from one brain to allow digestion by chondroitinase expressed in the sample. They were then stained for CS. Top row: incubated with extract of control brain (0), and with various amounts of commercial chondroitinase (12.5–100 mU). Bottom panels: incubated with extracts of LV-injected brains. All samples from the injected side gave some digestion, especially the 4-week samples, as did the 4-week samples from the opposite side. (j,k) 2B6 staining on spinal cord after LV-P cortical injection. (j) 2 weeks: horizontal section at level C3-C4. (k) 4 weeks: transverse section at level C1. M, midline. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Journal of Neuroscience Methods

    Article Title: Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

    doi: 10.1016/j.jneumeth.2011.08.003

    Figure Lengend Snippet: In vivo LV-ChABC injection into the rat cortex results in chondroitinase activity and the activity is also transported along axons. Injection was into deep layers of cortex on the left. Scale bar, 1 mm. (a–c) LV-C, 2 weeks: adjacent coronal sections from one brain stained for chondroitinase-produced stub (a), perineuronal net (b) and chondroitin sulfate (c). Chondroitinase digestion (area outlined with red arrows) is seen around the injection site and in the cortical white matter and adjacent hippocampus. On the original of (a), a faint column of reactivity can also be seen in the contralateral cortex. M, midline. (d–f) LV-C, 4 weeks: a similar series of sections, showing that chondroitinase digestion has extended along axon tracts and to the contralateral cortex. (g,h) LV-GFP, 4 weeks: similar sections from a brain injected with a lentivector encoding farnesyl-GFP; 4 weeks; stained with antibody against GFP. (g) Cortex near injection site, showing strong expression in astrocytes at surface (A), and oligodendrocytes in white matter/corpus callosum (O), and large numbers of neuronal fibers (N). (h) High-power views of contralateral side showing GFP-positive axons that have crossed in the corpus callosum and (inset) ascending in the cortex opposite the injection site. (i) Enzyme activity assay of functional chondroitinase level 4 weeks after LV injection into the cortex. Disks were loaded with equal amounts of chondroitin sulfate (CS) and each was incubated with extract of cortex from one brain to allow digestion by chondroitinase expressed in the sample. They were then stained for CS. Top row: incubated with extract of control brain (0), and with various amounts of commercial chondroitinase (12.5–100 mU). Bottom panels: incubated with extracts of LV-injected brains. All samples from the injected side gave some digestion, especially the 4-week samples, as did the 4-week samples from the opposite side. (j,k) 2B6 staining on spinal cord after LV-P cortical injection. (j) 2 weeks: horizontal section at level C3-C4. (k) 4 weeks: transverse section at level C1. M, midline. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: The antibody against chondroitinase ABC was kindly given by Acorda Inc.

    Techniques: In Vivo, Injection, Activity Assay, Staining, Produced, Expressing, Enzyme Activity Assay, Functional Assay, Incubation

    Western blots showing that active chondroitinase is secreted following transduction of tissue culture cells with the lentiviral vectors. Neu7 conditioned medium (a source of CSPGs) was placed on transfected cells for 24 h, then analyzed by western blotting. (a) LV-C and LV-D. The first two lanes are positive and negative controls with Neu7 medium not exposed to transfected cells; lane 1 was digested in vitro with commercial chondroitinase (Sigma, 20 mU, 37°, 3 h). (Top panel) Probed for carbohydrate ‘stub’ epitope produced by chondroitinase action (antibody 1B5). Neu7 conditioned medium (lane 2) shows little immunoreactivity, but incubation with cells after transduction with LV-ChABCs generates extensive reactivity. (Middle) Probed for NG2. Undigested NG2 (lane 2) appears largely as a characteristic ‘smear’ above the core protein band due to the heterodispersed high-Mr GAG chains, and this is all converted to core protein by digestion with commercial chondroitinase (lane 1) or by incubation with cells after transduction with LV-ChABC. (Bottom) Probed for chondroitinase ABC. Commercial chondroitinase (lane 1) shows both full-length band (Ch) and a shorter band (Ch**) due to proteolytic activity during incubation with medium. LV-ChABC all generate a diffuse chondroitinase band (GlyCh: partially glycosylated): this migrates more slowly than bacterial chondroitinase, confirming that it has some glycosylation at sites which were not mutated. Some lanes are overexposed as the experiment was principally intended to detect digestion of CSPGs; also see (c). Black bar or star indicates intense bands which partially bleached during imaging. (b) LV-GFP (control) and LV-P. (Top) Probed for NG2; the high-Mr smear due to GAG chains (marked NG2:CSPG), whose distribution varies according to cell type, is all reduced to core protein following LV-P transduction. (Bottom) Probed for chondroitinase ABC. (c) Confirmation of chondroitinase secretion. (1) Commercial chondroitinase (Sigma); (2) medium from HEK cells transduced with LV-C; (3) the same after treatment with N -glycosidase to remove residual glycosylation.

    Journal: Journal of Neuroscience Methods

    Article Title: Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

    doi: 10.1016/j.jneumeth.2011.08.003

    Figure Lengend Snippet: Western blots showing that active chondroitinase is secreted following transduction of tissue culture cells with the lentiviral vectors. Neu7 conditioned medium (a source of CSPGs) was placed on transfected cells for 24 h, then analyzed by western blotting. (a) LV-C and LV-D. The first two lanes are positive and negative controls with Neu7 medium not exposed to transfected cells; lane 1 was digested in vitro with commercial chondroitinase (Sigma, 20 mU, 37°, 3 h). (Top panel) Probed for carbohydrate ‘stub’ epitope produced by chondroitinase action (antibody 1B5). Neu7 conditioned medium (lane 2) shows little immunoreactivity, but incubation with cells after transduction with LV-ChABCs generates extensive reactivity. (Middle) Probed for NG2. Undigested NG2 (lane 2) appears largely as a characteristic ‘smear’ above the core protein band due to the heterodispersed high-Mr GAG chains, and this is all converted to core protein by digestion with commercial chondroitinase (lane 1) or by incubation with cells after transduction with LV-ChABC. (Bottom) Probed for chondroitinase ABC. Commercial chondroitinase (lane 1) shows both full-length band (Ch) and a shorter band (Ch**) due to proteolytic activity during incubation with medium. LV-ChABC all generate a diffuse chondroitinase band (GlyCh: partially glycosylated): this migrates more slowly than bacterial chondroitinase, confirming that it has some glycosylation at sites which were not mutated. Some lanes are overexposed as the experiment was principally intended to detect digestion of CSPGs; also see (c). Black bar or star indicates intense bands which partially bleached during imaging. (b) LV-GFP (control) and LV-P. (Top) Probed for NG2; the high-Mr smear due to GAG chains (marked NG2:CSPG), whose distribution varies according to cell type, is all reduced to core protein following LV-P transduction. (Bottom) Probed for chondroitinase ABC. (c) Confirmation of chondroitinase secretion. (1) Commercial chondroitinase (Sigma); (2) medium from HEK cells transduced with LV-C; (3) the same after treatment with N -glycosidase to remove residual glycosylation.

    Article Snippet: The antibody against chondroitinase ABC was kindly given by Acorda Inc.

    Techniques: Western Blot, Transduction, Transfection, In Vitro, Produced, Incubation, Activity Assay, Imaging