full-length syndecan core proteins Search Results


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  • 93
    BioVendor Instruments adiponectin mouse elisa
    Adiponectin Mouse Elisa, supplied by BioVendor Instruments, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological syndecan 4
    Syndecan 4, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    4Gene full length syndecan 4 gene
    In vitro migration and wound closure is increased by FGF-2 treatment in combination with <t>syndecan-4</t> proteoliposomes. Confluent monolayers of endothelial cells were wounded by scraping with a plastic cell scraper. Wound closure and cell migration was examined
    Full Length Syndecan 4 Gene, supplied by 4Gene, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore insulin
    In vitro migration and wound closure is increased by FGF-2 treatment in combination with <t>syndecan-4</t> proteoliposomes. Confluent monolayers of endothelial cells were wounded by scraping with a plastic cell scraper. Wound closure and cell migration was examined
    Insulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery syndecan 4
    In vitro migration and wound closure is increased by FGF-2 treatment in combination with <t>syndecan-4</t> proteoliposomes. Confluent monolayers of endothelial cells were wounded by scraping with a plastic cell scraper. Wound closure and cell migration was examined
    Syndecan 4, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies anti ha antibody
    In vitro migration and wound closure is increased by FGF-2 treatment in combination with <t>syndecan-4</t> proteoliposomes. Confluent monolayers of endothelial cells were wounded by scraping with a plastic cell scraper. Wound closure and cell migration was examined
    Anti Ha Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    AMS Biotechnology heparitinase
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not <t>heparitinase-treated</t> syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
    Heparitinase, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore immobilon p membrane
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not <t>heparitinase-treated</t> syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
    Immobilon P Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14510 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AvesLabs chicken polyclonal antibody
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not <t>heparitinase-treated</t> syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
    Chicken Polyclonal Antibody, supplied by AvesLabs, used in various techniques. Bioz Stars score: 90/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies horseradish peroxidase conjugated goat anti mouse antibody
    Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not <t>heparitinase-treated</t> syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
    Horseradish Peroxidase Conjugated Goat Anti Mouse Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 76 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
    Chondroitinase Abc, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rat syndecan 1 core proteins
    The conserved phenylalanine in the syndecan transmembrane domain enhances the SDS-resistant formation of syndecan heterodimers. A , amino acid sequences of the rat <t>syndecan-1,</t> -2, -3, and -4 transmembrane domains. The conserved G XXX G motif is underlined
    Rat Syndecan 1 Core Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery rat sulf2 sirna
    Endogenous <t>SULF2</t> strongly inhibits the catabolism of model remnant lipoproteins by cultured liver cells. McArdle hepatoma cells were pre-incubated for 24h with 100nM non-target ( Control ) or Sulf2 <t>siRNA,</t> as indicated, followed by an additional 44h at 37°C. Cells were then incubated for 4h at 37°C with model remnant lipoproteins that we prepared by combining 125 I-labeled methylated LDL with lipoprotein lipase ( LpL ), a molecule that bridges between lipoproteins and HSPGs (background values were assessed in the absence of LpL). Panel A : Immunoblots of cellular homogenates, using anti-SULF2 antibodies or, as a loading control, anti-ß-actin antibodies. Panel B : LpL-dependent cellular catabolism of model remnant lipoproteins, shown as surface binding (Surf) , internalization (Inter) , and degradation (Degr) , normalized to control values from cells treated with the nontarget siRNA (means±SEMs, n=3; the non-normalized control values were 194±2.9, 605±20.6, and 112±4.3 ng/mg, respectively). **, P
    Rat Sulf2 Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology syndecan 1 core protein
    Endogenous <t>SULF2</t> strongly inhibits the catabolism of model remnant lipoproteins by cultured liver cells. McArdle hepatoma cells were pre-incubated for 24h with 100nM non-target ( Control ) or Sulf2 <t>siRNA,</t> as indicated, followed by an additional 44h at 37°C. Cells were then incubated for 4h at 37°C with model remnant lipoproteins that we prepared by combining 125 I-labeled methylated LDL with lipoprotein lipase ( LpL ), a molecule that bridges between lipoproteins and HSPGs (background values were assessed in the absence of LpL). Panel A : Immunoblots of cellular homogenates, using anti-SULF2 antibodies or, as a loading control, anti-ß-actin antibodies. Panel B : LpL-dependent cellular catabolism of model remnant lipoproteins, shown as surface binding (Surf) , internalization (Inter) , and degradation (Degr) , normalized to control values from cells treated with the nontarget siRNA (means±SEMs, n=3; the non-normalized control values were 194±2.9, 605±20.6, and 112±4.3 ng/mg, respectively). **, P
    Syndecan 1 Core Protein, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher syndecan 2 ectodomain coding sequence
    The <t>syndecan-2</t> extracellular core protein inhibits angiogenesis. (A) Schematic diagram of S2ED consisting of GST fused to the syndecan-2 <t>ectodomain</t> core protein. (B) Angiogenesis is inhibited by S2ED when incorporated into Matrigel. C57BL/6 mice were given sub-dermal injections of Matrigel supplemented with VEGF and FGF containing either 0.5 µM of S2ED or GST. Excised sections of Matrigel plugs were immunostained for CD31 (endothelial cells) and DRAQ5 (all cell nuclei) and analysed by confocal microscopy, and the percentage of cells positive for CD31 calculated. Less endothelial cells migrate into Matrigel plugs containing S2ED. Error bars represent the s.e.m. of four separate images. * P
    Syndecan 2 Ectodomain Coding Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    4Gene syndecan 4
    Engagement of <t>syndecan-4</t> determines both the direction and persistence of migration. (A) Syndecan-4 limits Rac1 activity in the absence of matrix engagement and induces activation in response to fibronectin. (B and C) By constraining localized Rac1 activation to developing points of contact with the ECM (red), syndecan-4 coordinates migration along a fibronectin fibril. Consequently, wild-type fibroblasts migrate persistently over a meshwork of similar fibers (B) but follow the dominant strand when presented with a choice of paths (C). In contrast, syndecan-4–null cells protrude in multiple directions, rendering progression inefficient, whereas mutants in the PKCα-binding motif fail to respond to changes in matrix organization or follow the optimal path.
    Syndecan 4, supplied by 4Gene, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam syndecan 4
    Measurement of <t>syndecan-4</t> levels in the skin of diabetic and healthy patients; synthesis and characterization of syndesomes
    Syndecan 4, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen ni nta agarose columns
    Measurement of <t>syndecan-4</t> levels in the skin of diabetic and healthy patients; synthesis and characterization of syndesomes
    Ni Nta Agarose Columns, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vitro migration and wound closure is increased by FGF-2 treatment in combination with syndecan-4 proteoliposomes. Confluent monolayers of endothelial cells were wounded by scraping with a plastic cell scraper. Wound closure and cell migration was examined

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle

    doi: 10.1073/pnas.1117885109

    Figure Lengend Snippet: In vitro migration and wound closure is increased by FGF-2 treatment in combination with syndecan-4 proteoliposomes. Confluent monolayers of endothelial cells were wounded by scraping with a plastic cell scraper. Wound closure and cell migration was examined

    Article Snippet: A constitutive expression vector containing the full-length syndecan-4 gene (Origene; National Center for Biotechnology Information Reference Sequence ) was transfected into HeLa cells using the FuGENE HD transfection reagent (Roche) per the manufacturer's specifications.

    Techniques: In Vitro, Migration

    Syndecan-4 proteoliposomes enhance FGF-2–induced proliferation, migration, and intracellular signaling. ( A ) Syndecan-4 proteoliposomes induce enhanced endothelial proliferation in response to FGF-2 treatment. Black bars are samples without FGF-2

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle

    doi: 10.1073/pnas.1117885109

    Figure Lengend Snippet: Syndecan-4 proteoliposomes enhance FGF-2–induced proliferation, migration, and intracellular signaling. ( A ) Syndecan-4 proteoliposomes induce enhanced endothelial proliferation in response to FGF-2 treatment. Black bars are samples without FGF-2

    Article Snippet: A constitutive expression vector containing the full-length syndecan-4 gene (Origene; National Center for Biotechnology Information Reference Sequence ) was transfected into HeLa cells using the FuGENE HD transfection reagent (Roche) per the manufacturer's specifications.

    Techniques: Migration

    Syndecan-4 proteoliposomes enhance FGF-2 uptake and intracellular trafficking. ( A ) Liposomally embedded syndecan-4 increases uptake of 125 I-labeled FGF-2 in endothelial cells. FGF-2 alone (blue line), FGF-2 with liposomes (green line), FGF-2 with syndecan-4

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle

    doi: 10.1073/pnas.1117885109

    Figure Lengend Snippet: Syndecan-4 proteoliposomes enhance FGF-2 uptake and intracellular trafficking. ( A ) Liposomally embedded syndecan-4 increases uptake of 125 I-labeled FGF-2 in endothelial cells. FGF-2 alone (blue line), FGF-2 with liposomes (green line), FGF-2 with syndecan-4

    Article Snippet: A constitutive expression vector containing the full-length syndecan-4 gene (Origene; National Center for Biotechnology Information Reference Sequence ) was transfected into HeLa cells using the FuGENE HD transfection reagent (Roche) per the manufacturer's specifications.

    Techniques: Labeling

    Enhanced neovascularization of ischemic muscle after treatment with syndecan-4 proteoliposomes in combination with FGF-2. Hind-limb ischemia was created in rats through femoral artery ligation, and treatments were delivered over 7 d with an osmotic pump.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle

    doi: 10.1073/pnas.1117885109

    Figure Lengend Snippet: Enhanced neovascularization of ischemic muscle after treatment with syndecan-4 proteoliposomes in combination with FGF-2. Hind-limb ischemia was created in rats through femoral artery ligation, and treatments were delivered over 7 d with an osmotic pump.

    Article Snippet: A constitutive expression vector containing the full-length syndecan-4 gene (Origene; National Center for Biotechnology Information Reference Sequence ) was transfected into HeLa cells using the FuGENE HD transfection reagent (Roche) per the manufacturer's specifications.

    Techniques: Ligation

    Syndecan-4 proteoliposomes enhance in vitro tube formation in combination with FGF-2. HUVECs were seeded on extracellular matrix and exposed to various treatments. In vitro tube formation was quantified by measuring total tube length and the number of

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle

    doi: 10.1073/pnas.1117885109

    Figure Lengend Snippet: Syndecan-4 proteoliposomes enhance in vitro tube formation in combination with FGF-2. HUVECs were seeded on extracellular matrix and exposed to various treatments. In vitro tube formation was quantified by measuring total tube length and the number of

    Article Snippet: A constitutive expression vector containing the full-length syndecan-4 gene (Origene; National Center for Biotechnology Information Reference Sequence ) was transfected into HeLa cells using the FuGENE HD transfection reagent (Roche) per the manufacturer's specifications.

    Techniques: In Vitro

    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

    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

    The conserved phenylalanine in the syndecan transmembrane domain enhances the SDS-resistant formation of syndecan heterodimers. A , amino acid sequences of the rat syndecan-1, -2, -3, and -4 transmembrane domains. The conserved G XXX G motif is underlined

    Journal: The Journal of Biological Chemistry

    Article Title: The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans *

    doi: 10.1074/jbc.M115.685040

    Figure Lengend Snippet: The conserved phenylalanine in the syndecan transmembrane domain enhances the SDS-resistant formation of syndecan heterodimers. A , amino acid sequences of the rat syndecan-1, -2, -3, and -4 transmembrane domains. The conserved G XXX G motif is underlined

    Article Snippet: The cDNAs encoding the full-length rat syndecan-2 or -4 core proteins, the single point mutants (S2(F167Y), S2(F169Y), S2(F172Y), and S4(F164Y)), rat syndecan-1 core proteins, and the point mutants (S1(F269Y), S1(F269Y,C272F), and S1(C272F)), and rat syndecan-3 core proteins were synthesized by PCR and subcloned into the His-tagging expression vector pET32a+ (Novagen, Madison, WI).

    Techniques:

    The conserved phenylalanine has a critical role in syndecan-1 heterodimer formation. A , B , and E , the indicated syndecan wild type and mutants were mixed for 10 min on ice, separated by 8% SDS-PAGE, and stained with Coomassie Blue. The data shown are

    Journal: The Journal of Biological Chemistry

    Article Title: The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans *

    doi: 10.1074/jbc.M115.685040

    Figure Lengend Snippet: The conserved phenylalanine has a critical role in syndecan-1 heterodimer formation. A , B , and E , the indicated syndecan wild type and mutants were mixed for 10 min on ice, separated by 8% SDS-PAGE, and stained with Coomassie Blue. The data shown are

    Article Snippet: The cDNAs encoding the full-length rat syndecan-2 or -4 core proteins, the single point mutants (S2(F167Y), S2(F169Y), S2(F172Y), and S4(F164Y)), rat syndecan-1 core proteins, and the point mutants (S1(F269Y), S1(F269Y,C272F), and S1(C272F)), and rat syndecan-3 core proteins were synthesized by PCR and subcloned into the His-tagging expression vector pET32a+ (Novagen, Madison, WI).

    Techniques: SDS Page, Staining

    Endogenous SULF2 strongly inhibits the catabolism of model remnant lipoproteins by cultured liver cells. McArdle hepatoma cells were pre-incubated for 24h with 100nM non-target ( Control ) or Sulf2 siRNA, as indicated, followed by an additional 44h at 37°C. Cells were then incubated for 4h at 37°C with model remnant lipoproteins that we prepared by combining 125 I-labeled methylated LDL with lipoprotein lipase ( LpL ), a molecule that bridges between lipoproteins and HSPGs (background values were assessed in the absence of LpL). Panel A : Immunoblots of cellular homogenates, using anti-SULF2 antibodies or, as a loading control, anti-ß-actin antibodies. Panel B : LpL-dependent cellular catabolism of model remnant lipoproteins, shown as surface binding (Surf) , internalization (Inter) , and degradation (Degr) , normalized to control values from cells treated with the nontarget siRNA (means±SEMs, n=3; the non-normalized control values were 194±2.9, 605±20.6, and 112±4.3 ng/mg, respectively). **, P

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Type 2 Diabetes in Mice Induces Hepatic Overexpression of Sulfatase 2, a Novel Factor That Suppresses Uptake of Remnant Lipoproteins

    doi: 10.1002/hep.23916

    Figure Lengend Snippet: Endogenous SULF2 strongly inhibits the catabolism of model remnant lipoproteins by cultured liver cells. McArdle hepatoma cells were pre-incubated for 24h with 100nM non-target ( Control ) or Sulf2 siRNA, as indicated, followed by an additional 44h at 37°C. Cells were then incubated for 4h at 37°C with model remnant lipoproteins that we prepared by combining 125 I-labeled methylated LDL with lipoprotein lipase ( LpL ), a molecule that bridges between lipoproteins and HSPGs (background values were assessed in the absence of LpL). Panel A : Immunoblots of cellular homogenates, using anti-SULF2 antibodies or, as a loading control, anti-ß-actin antibodies. Panel B : LpL-dependent cellular catabolism of model remnant lipoproteins, shown as surface binding (Surf) , internalization (Inter) , and degradation (Degr) , normalized to control values from cells treated with the nontarget siRNA (means±SEMs, n=3; the non-normalized control values were 194±2.9, 605±20.6, and 112±4.3 ng/mg, respectively). **, P

    Article Snippet: Antibodies against SULF2 and the syndecan-1 core protein (catalogue #sc68436 and #sc-5632, Santa Cruz Biotechnology, Santa Cruz, CA), rat Sulf2 siRNA (#M-093673-00-0010, Smartpool, Dharmacon, Lafayette, CO), control siRNA, ELISA kits to quantify mouse adiponectin (#RD293023100R, BioVendor, Candler, NC) and insulin (#EZRMI-13K, Millipore, Billerica, MA), full-length adiponectin prepared in eukaryotic cells (fAd, Pepro Technology, Rocky Hill, NJ), and insulin (#I2643, Sigma Chemical Company, St. Louis, MO) were obtained commercially.

    Techniques: Cell Culture, Incubation, Labeling, Methylation, Western Blot, Binding Assay

    The syndecan-2 extracellular core protein inhibits angiogenesis. (A) Schematic diagram of S2ED consisting of GST fused to the syndecan-2 ectodomain core protein. (B) Angiogenesis is inhibited by S2ED when incorporated into Matrigel. C57BL/6 mice were given sub-dermal injections of Matrigel supplemented with VEGF and FGF containing either 0.5 µM of S2ED or GST. Excised sections of Matrigel plugs were immunostained for CD31 (endothelial cells) and DRAQ5 (all cell nuclei) and analysed by confocal microscopy, and the percentage of cells positive for CD31 calculated. Less endothelial cells migrate into Matrigel plugs containing S2ED. Error bars represent the s.e.m. of four separate images. * P

    Journal: Journal of Cell Science

    Article Title: Shed syndecan-2 inhibits angiogenesis

    doi: 10.1242/jcs.153015

    Figure Lengend Snippet: The syndecan-2 extracellular core protein inhibits angiogenesis. (A) Schematic diagram of S2ED consisting of GST fused to the syndecan-2 ectodomain core protein. (B) Angiogenesis is inhibited by S2ED when incorporated into Matrigel. C57BL/6 mice were given sub-dermal injections of Matrigel supplemented with VEGF and FGF containing either 0.5 µM of S2ED or GST. Excised sections of Matrigel plugs were immunostained for CD31 (endothelial cells) and DRAQ5 (all cell nuclei) and analysed by confocal microscopy, and the percentage of cells positive for CD31 calculated. Less endothelial cells migrate into Matrigel plugs containing S2ED. Error bars represent the s.e.m. of four separate images. * P

    Article Snippet: Generation of syndecan-2-expressing cell lines Gene synthesis of the complete murine syndecan-2 cDNA and the cDNA encoding only the syndecan-2 ectodomain coding sequence was performed by GeneArt (Invitrogen).

    Techniques: Mouse Assay, Confocal Microscopy

    Engagement of syndecan-4 determines both the direction and persistence of migration. (A) Syndecan-4 limits Rac1 activity in the absence of matrix engagement and induces activation in response to fibronectin. (B and C) By constraining localized Rac1 activation to developing points of contact with the ECM (red), syndecan-4 coordinates migration along a fibronectin fibril. Consequently, wild-type fibroblasts migrate persistently over a meshwork of similar fibers (B) but follow the dominant strand when presented with a choice of paths (C). In contrast, syndecan-4–null cells protrude in multiple directions, rendering progression inefficient, whereas mutants in the PKCα-binding motif fail to respond to changes in matrix organization or follow the optimal path.

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: Engagement of syndecan-4 determines both the direction and persistence of migration. (A) Syndecan-4 limits Rac1 activity in the absence of matrix engagement and induces activation in response to fibronectin. (B and C) By constraining localized Rac1 activation to developing points of contact with the ECM (red), syndecan-4 coordinates migration along a fibronectin fibril. Consequently, wild-type fibroblasts migrate persistently over a meshwork of similar fibers (B) but follow the dominant strand when presented with a choice of paths (C). In contrast, syndecan-4–null cells protrude in multiple directions, rendering progression inefficient, whereas mutants in the PKCα-binding motif fail to respond to changes in matrix organization or follow the optimal path.

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Migration, Activity Assay, Activation Assay, Binding Assay

    Expression of syndecan-4 determines the persistence of migration on cell-derived matrices. (A) Cell-derived matrices were generated by culturing human fibroblasts for 8 d before denuding the confluent fibroblasts and reseeding mutant MEF lines. (B) MEFs were seeded onto cell-derived matrices and were allowed to grow for 8 h before filming for 10 h. Persistence was determined by dividing the linear displacement of a cell by total distance migrated. Gray blocks represent the experimentally determined threshold for the random migration of cells on fibronectin-coated glass. (C–F) Migration tracks of MEFs over the 10-h filming period. The tracks of cells from three different fields of view have been compressed into each panel. (G) The number of lamellae present in syndecan-4–null MEFs (gray), reexpressing wild-type (black), or Y188L mutant (white) syndecan-4 were scored manually in all tracked cells at a single time point. Error bars indicate the SEM of 30 different cells, and asterisks indicate a significant difference in persistence (*, P

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: Expression of syndecan-4 determines the persistence of migration on cell-derived matrices. (A) Cell-derived matrices were generated by culturing human fibroblasts for 8 d before denuding the confluent fibroblasts and reseeding mutant MEF lines. (B) MEFs were seeded onto cell-derived matrices and were allowed to grow for 8 h before filming for 10 h. Persistence was determined by dividing the linear displacement of a cell by total distance migrated. Gray blocks represent the experimentally determined threshold for the random migration of cells on fibronectin-coated glass. (C–F) Migration tracks of MEFs over the 10-h filming period. The tracks of cells from three different fields of view have been compressed into each panel. (G) The number of lamellae present in syndecan-4–null MEFs (gray), reexpressing wild-type (black), or Y188L mutant (white) syndecan-4 were scored manually in all tracked cells at a single time point. Error bars indicate the SEM of 30 different cells, and asterisks indicate a significant difference in persistence (*, P

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Expressing, Migration, Derivative Assay, Generated, Mutagenesis

    Expression of syndecan-4 regulates the localized activity of Rac1. (A) MEF lines were allowed to spread on cell-derived matrices for 4 h before GTP-Rac1 levels were measured using effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. Error bars indicate SEM, and asterisks indicate significant activation (*, P

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: Expression of syndecan-4 regulates the localized activity of Rac1. (A) MEF lines were allowed to spread on cell-derived matrices for 4 h before GTP-Rac1 levels were measured using effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. Error bars indicate SEM, and asterisks indicate significant activation (*, P

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Expressing, Activity Assay, Derivative Assay, Western Blot, Activation Assay

    The PKCα-binding motif of syndecan-4 mediates Rac1 regulation and adhesion complex formation. (A) Schematic representation of the syndecan-4 cytoplasmic domain. Tyr-188 is a key element of the PKCα-binding motif ( Lim et al., 2003 ), and Tyr-180 was chosen as a negative control. (B and C) Syndecan-4–null MEFs expressing Y188L (B) or Y180L (C) mutant cDNAs were plated onto fibronectin, and GTP-Rac1 levels were measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. (D) Relative levels of GTP-Rac1 in fully spread cells were compared between lines. Equivalent loading between experiments was confirmed by blotting crude lysates for total Rac1 and vinculin. Error bars indicate SEM, and asterisks indicate significant activation (P

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: The PKCα-binding motif of syndecan-4 mediates Rac1 regulation and adhesion complex formation. (A) Schematic representation of the syndecan-4 cytoplasmic domain. Tyr-188 is a key element of the PKCα-binding motif ( Lim et al., 2003 ), and Tyr-180 was chosen as a negative control. (B and C) Syndecan-4–null MEFs expressing Y188L (B) or Y180L (C) mutant cDNAs were plated onto fibronectin, and GTP-Rac1 levels were measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. (D) Relative levels of GTP-Rac1 in fully spread cells were compared between lines. Equivalent loading between experiments was confirmed by blotting crude lysates for total Rac1 and vinculin. Error bars indicate SEM, and asterisks indicate significant activation (P

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Binding Assay, Negative Control, Expressing, Mutagenesis, Western Blot, Activation Assay

    Engagement of syndecan-4 drives the biphasic formation of adhesion complexes. The processes of spreading and adhesion complex formation were followed by staining fixed cells for vinculin and actin and measuring the cell area (A and C) or focal adhesion area (B and H) of 100 cells or the mean focal adhesion length (I) of 30 cells using ImageJ software. (A and B) Primary fibroblasts spreading on 50K (circles) or fibronectin (crosses). (C) Wild-type (crosses), syndecan-4–null (circles), or rescued (squares) MEFs spreading on fibronectin. (D–G) Adhesion complex formation in response to syndecan-4 engagement was followed in primary fibroblasts prespread on 50K for 2 h before stimulation with H/0 (D), a nonheparin-binding mutant of H/0 (E), nonimmune IgG (F), or 5G9 monoclonal antibody directed against the syndecan-4 extracellular domain (G). (H and I) Focal adhesion area (H) and mean focal contact length (I) of primary fibroblasts prespread on 50K for 2 h before stimulation with syndecan-4 ligands. Images and analyses are representative of experiments performed on six separate occasions. Error bars indicate SEM. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: Engagement of syndecan-4 drives the biphasic formation of adhesion complexes. The processes of spreading and adhesion complex formation were followed by staining fixed cells for vinculin and actin and measuring the cell area (A and C) or focal adhesion area (B and H) of 100 cells or the mean focal adhesion length (I) of 30 cells using ImageJ software. (A and B) Primary fibroblasts spreading on 50K (circles) or fibronectin (crosses). (C) Wild-type (crosses), syndecan-4–null (circles), or rescued (squares) MEFs spreading on fibronectin. (D–G) Adhesion complex formation in response to syndecan-4 engagement was followed in primary fibroblasts prespread on 50K for 2 h before stimulation with H/0 (D), a nonheparin-binding mutant of H/0 (E), nonimmune IgG (F), or 5G9 monoclonal antibody directed against the syndecan-4 extracellular domain (G). (H and I) Focal adhesion area (H) and mean focal contact length (I) of primary fibroblasts prespread on 50K for 2 h before stimulation with syndecan-4 ligands. Images and analyses are representative of experiments performed on six separate occasions. Error bars indicate SEM. Bar, 10 μm.

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Staining, Software, Binding Assay, Mutagenesis

    Engagement of syndecan-4 is essential for activation of Rac1 during adhesion to fibronectin. GTP-Rac1 levels during cell spreading or in response to H/0 were measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. (A and B) Primary human fibroblasts were plated onto fibronectin (A) or 50K (B), and lysates were prepared after appropriate time periods. (C–E) The necessity of syndecan-4 expression for Rac1 regulation during spreading on fibronectin was tested using wild-type (C), syndecan-4–null (D), or syndecan-4–null transfected with full-length syndecan-4 cDNA MEFs (E). (F) Relative levels of GTP-Rac1 were directly compared between cell lines either fully spread (120 min) or during spreading on fibronectin for 60 min. (G) Rac1 activation in response to soluble H/0 in primary fibroblasts prespread on 50K. Equivalent loading between experiments was confirmed by blotting crude lysates for total Rac1 and vinculin. Axes are given in arbitrary units assigned according to the relative activity of fully spread cell lines. Each panel is representative of at least four separate experiments, and error bars indicate SEM. Asterisks indicate significant activation (*, P

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: Engagement of syndecan-4 is essential for activation of Rac1 during adhesion to fibronectin. GTP-Rac1 levels during cell spreading or in response to H/0 were measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. (A and B) Primary human fibroblasts were plated onto fibronectin (A) or 50K (B), and lysates were prepared after appropriate time periods. (C–E) The necessity of syndecan-4 expression for Rac1 regulation during spreading on fibronectin was tested using wild-type (C), syndecan-4–null (D), or syndecan-4–null transfected with full-length syndecan-4 cDNA MEFs (E). (F) Relative levels of GTP-Rac1 were directly compared between cell lines either fully spread (120 min) or during spreading on fibronectin for 60 min. (G) Rac1 activation in response to soluble H/0 in primary fibroblasts prespread on 50K. Equivalent loading between experiments was confirmed by blotting crude lysates for total Rac1 and vinculin. Axes are given in arbitrary units assigned according to the relative activity of fully spread cell lines. Each panel is representative of at least four separate experiments, and error bars indicate SEM. Asterisks indicate significant activation (*, P

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Activation Assay, Western Blot, Expressing, Transfection, Activity Assay

    Engagement of syndecan-4 contributes toward but is not essential for the regulation of RhoA during adhesion to fibronectin. GTPase activity was measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. (A and B) Primary fibroblasts were prespread on 50K for 2 h before measuring the activity of Cdc42 (A) or RhoA (B) in response to stimulation with H/0. (C and D) RhoA activity was measured during spreading on fibronectin (C) or 50K (D). Equivalent loading between experiments was confirmed by blotting crude lysates for total GTPase and vinculin. Each panel is representative of at least four separate experiments, and error bars indicate SEM. Asterisks indicate significant activation (P

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: Engagement of syndecan-4 contributes toward but is not essential for the regulation of RhoA during adhesion to fibronectin. GTPase activity was measured by effector pull-down assays in combination with quantitative Western blotting using fluorophore-conjugated antibodies. (A and B) Primary fibroblasts were prespread on 50K for 2 h before measuring the activity of Cdc42 (A) or RhoA (B) in response to stimulation with H/0. (C and D) RhoA activity was measured during spreading on fibronectin (C) or 50K (D). Equivalent loading between experiments was confirmed by blotting crude lysates for total GTPase and vinculin. Each panel is representative of at least four separate experiments, and error bars indicate SEM. Asterisks indicate significant activation (P

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Activity Assay, Western Blot, Activation Assay

    The PKCα-binding motif of syndecan-4 allows cells to turn in response to changes in the matrix environment. (A and B) MEFs were seeded onto fibronectin-coated gold stripes (A) and allowed to spread for 2 h before filming for 10 h and classifying turns as direct (retaining contact with the apex), indirect (forced by collision with the opposing wall), or no turn (B). (C–J) MEFs rescued with wild-type (C and F) or Y188L (D and G) syndecan-4 were tracked moving past a branch point with the option of turning (C and D) or toward a corner, where they were forced to turn (F and G). Cells migrating along gold stripes coated with fibronectin (E and H) or vitronectin (I and J) were scored for the ability to make direct, indirect, or no turn as they migrated past a junction (E and I) or toward a corner (H and J). 60 cells expressing wild-type (black bars) or Y188L mutant (white bars) syndecan-4 were tracked for each condition, and the experiment was repeated on three separate occasions. Error bars indicate the SEM, and asterisks indicate a significant difference (*, P

    Journal: The Journal of Cell Biology

    Article Title: Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    doi: 10.1083/jcb.200610076

    Figure Lengend Snippet: The PKCα-binding motif of syndecan-4 allows cells to turn in response to changes in the matrix environment. (A and B) MEFs were seeded onto fibronectin-coated gold stripes (A) and allowed to spread for 2 h before filming for 10 h and classifying turns as direct (retaining contact with the apex), indirect (forced by collision with the opposing wall), or no turn (B). (C–J) MEFs rescued with wild-type (C and F) or Y188L (D and G) syndecan-4 were tracked moving past a branch point with the option of turning (C and D) or toward a corner, where they were forced to turn (F and G). Cells migrating along gold stripes coated with fibronectin (E and H) or vitronectin (I and J) were scored for the ability to make direct, indirect, or no turn as they migrated past a junction (E and I) or toward a corner (H and J). 60 cells expressing wild-type (black bars) or Y188L mutant (white bars) syndecan-4 were tracked for each condition, and the experiment was repeated on three separate occasions. Error bars indicate the SEM, and asterisks indicate a significant difference (*, P

    Article Snippet: The effect of syndecan-4 on the expression of other matrix receptors that might contribute toward Rac1 regulation was assessed by flow cytometric analysis and revealed that neither disruption nor reexpression of the syndecan-4 gene had any effect on the surface expression of syndecans-1 or -2 or the integrin α5 or β1 subunits (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200610076/DC1 ), thereby confirming the specific role for syndecan-4 in Rac1 regulation.

    Techniques: Binding Assay, Expressing, Mutagenesis

    Measurement of syndecan-4 levels in the skin of diabetic and healthy patients; synthesis and characterization of syndesomes

    Journal: Advanced healthcare materials

    Article Title: Syndesome Therapeutics for Enhancing Diabetic Wound Healing

    doi: 10.1002/adhm.201600285

    Figure Lengend Snippet: Measurement of syndecan-4 levels in the skin of diabetic and healthy patients; synthesis and characterization of syndesomes

    Article Snippet: The syndecan-4 overexpressing stable cells were grown in high glucose DMEM with 10% FBS, 5% Penicillin-Streptomycin and 5% L-glutamine.

    Techniques: