heparitinase Search Results


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  • 95
    New England Biolabs bacteroides heparinase iii
    Bacteroides Heparinase Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore heparinase iii
    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without <t>heparinase</t> <t>III</t> for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p
    Heparinase Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku heparitinase
    Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by <t>heparitinase</t> treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p
    Heparitinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    IBEX Technologies heparitinase
    HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with <t>heparitinase</t> to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.
    Heparitinase, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 91/100, based on 11 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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    93
    R&D Systems heparinase iii
    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and <t>Heparinase</t> <t>III.</t> Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Heparinase Iii, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Seikagaku mu heparinase iii
    Western blot of total L2 proteoglycans probed with affinity-purified R664 polyclonal antibodies against a fusion protein prepared using clone F15a (see Fig. 1 ). The proteoglycans were untreated (lane 1 ) or pretreated with <t>heparinase</t> <t>III</t> (lanes 2 and 3 ) and/or chondroitinase ABC (lanes 3 and 4 ). A core protein is only clearly resolved after removal of chondroitin sulfate chains. The migration of molecular mass markers in kD is shown.
    Mu Heparinase Iii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 80/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku heparitinase iii
    Western blot of total L2 proteoglycans probed with affinity-purified R664 polyclonal antibodies against a fusion protein prepared using clone F15a (see Fig. 1 ). The proteoglycans were untreated (lane 1 ) or pretreated with <t>heparinase</t> <t>III</t> (lanes 2 and 3 ) and/or chondroitinase ABC (lanes 3 and 4 ). A core protein is only clearly resolved after removal of chondroitin sulfate chains. The migration of molecular mass markers in kD is shown.
    Heparitinase Iii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore heparinase iii treatment
    GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or <t>Heparinase</t> <t>III</t> treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .
    Heparinase Iii Treatment, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ICN Biomedicals heparitinase
    The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of <t>heparitinase</t> treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.
    Heparitinase, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    IBEX Technologies iu heparinase iii
    En face and cross sections of RFPEC in which HS was immunostained in green and cell nuclei in blue. Notes: ( A ) Control, ( B ) low serum, ( C ) Hep <t>III,</t> and ( D ) Hep III + HS. ( E ) Thickness measurements of the glycocalyx when observing only the HS component by binding the 10E4 epitope HS antibody to the glycosaminoglycan under control, low serum, Hep III, and Hep III + HS treatments. HS thickness decreased significantly for all treatments. ( F ) HS coverage of RFPEC after various treatments. HS coverage decreased significantly after <t>heparinase</t> III treatment and recovered after HS addition. * P
    Iu Heparinase Iii, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    IBEX Technologies heparinase iii hep iii enzyme
    En face and cross sections of RFPEC in which HS was immunostained in green and cell nuclei in blue. Notes: ( A ) Control, ( B ) low serum, ( C ) Hep <t>III,</t> and ( D ) Hep III + HS. ( E ) Thickness measurements of the glycocalyx when observing only the HS component by binding the 10E4 epitope HS antibody to the glycosaminoglycan under control, low serum, Hep III, and Hep III + HS treatments. HS thickness decreased significantly for all treatments. ( F ) HS coverage of RFPEC after various treatments. HS coverage decreased significantly after <t>heparinase</t> III treatment and recovered after HS addition. * P
    Heparinase Iii Hep Iii Enzyme, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore heparitinase i
    En face and cross sections of RFPEC in which HS was immunostained in green and cell nuclei in blue. Notes: ( A ) Control, ( B ) low serum, ( C ) Hep <t>III,</t> and ( D ) Hep III + HS. ( E ) Thickness measurements of the glycocalyx when observing only the HS component by binding the 10E4 epitope HS antibody to the glycosaminoglycan under control, low serum, Hep III, and Hep III + HS treatments. HS thickness decreased significantly for all treatments. ( F ) HS coverage of RFPEC after various treatments. HS coverage decreased significantly after <t>heparinase</t> III treatment and recovered after HS addition. * P
    Heparitinase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Seikagaku flavobacterium heparinum heparitinase
    En face and cross sections of RFPEC in which HS was immunostained in green and cell nuclei in blue. Notes: ( A ) Control, ( B ) low serum, ( C ) Hep <t>III,</t> and ( D ) Hep III + HS. ( E ) Thickness measurements of the glycocalyx when observing only the HS component by binding the 10E4 epitope HS antibody to the glycosaminoglycan under control, low serum, Hep III, and Hep III + HS treatments. HS thickness decreased significantly for all treatments. ( F ) HS coverage of RFPEC after various treatments. HS coverage decreased significantly after <t>heparinase</t> III treatment and recovered after HS addition. * P
    Flavobacterium Heparinum Heparitinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    AMS Biotechnology miliunits heparinase iii
    Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by <t>heparinase</t> treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from <t>three</t> independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.
    Miliunits Heparinase Iii, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Flow Cytometry, DCFH-DA Assay, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in SH-SY5Y cell morphology and cell viability. ( a ) SH-SY5Y cell morphology. Bar, 10 μM. Images were analyzed with SPOT 4.7 Advanced software. The arrows indicate the shorter neurite outgrowth of SH-SY5Y cells after the Aβ(25-35) challenge. ( b ) SH-SY5Y cell viability was analyzed by an MTT assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in SH-SY5Y cell morphology and cell viability. ( a ) SH-SY5Y cell morphology. Bar, 10 μM. Images were analyzed with SPOT 4.7 Advanced software. The arrows indicate the shorter neurite outgrowth of SH-SY5Y cells after the Aβ(25-35) challenge. ( b ) SH-SY5Y cell viability was analyzed by an MTT assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Software, MTT Assay, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in the phosphorylated (p)-tau/tau ratio ( a ), phosphorylated (p)-phosphatidylinositol 3K (PI3K)/PI3K ratio ( b ), the phosphorylated (p)-Akt/Akt ratio ( c ), and the phosphorylated (p)-glycogen synthase kinase (GSK)-3β (Ser 9 )/GSK-3β ratio ( d ) of protein expressions. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in the phosphorylated (p)-tau/tau ratio ( a ), phosphorylated (p)-phosphatidylinositol 3K (PI3K)/PI3K ratio ( b ), the phosphorylated (p)-Akt/Akt ratio ( c ), and the phosphorylated (p)-glycogen synthase kinase (GSK)-3β (Ser 9 )/GSK-3β ratio ( d ) of protein expressions. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in cell apoptosis. ( a ) Western blot analysis of caspase-3 protein expression in SH-SY5Y cells. ( b ) Percentages of apoptotic cells in each group quantified from ( c ). ( c ) Representative profiles of cell apoptosis detected by flow cytometry with Annexin V/propidium iodide double-staining. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in cell apoptosis. ( a ) Western blot analysis of caspase-3 protein expression in SH-SY5Y cells. ( b ) Percentages of apoptotic cells in each group quantified from ( c ). ( c ) Representative profiles of cell apoptosis detected by flow cytometry with Annexin V/propidium iodide double-staining. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Western Blot, Expressing, Flow Cytometry, Double Staining, Incubation

    Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: IL-1 and TGF-β Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells

    doi: 10.1167/iovs.18-25202

    Figure Lengend Snippet: Regulation of EBM component protein expression by IL-1α and TGF-β1 in primary rabbit keratocytes. Primary keratocan+ keratocytes were cultured and treated with 10 ng/mL IL-1α, 2 ng/mL TGF-β1, or left untreated for 16 hours. Keratocytes to be used in the experiments were lysed and keratocan of the expected size (50 kDa 45 ) was detected (A) to confirm these cells were keratocan+ keratocytes at the beginning of the exposure. (B) Perlecan, (C) nidogen-1, and (D) nidogen-2 expression detected by Western blot. Cell extracts used for perlecan Western blots were treated with heparitinase III, as was described in the methods. β-actin was used as a loading control for each experiment. A representative Western blot of the three performed for each BM component is shown. The graphs beneath each Western blot was obtained by densitometry analysis of the bands from each of the three Western blots from different experiments. *The change in BM protein was statistically significant (P

    Article Snippet: For perlecan Western blots, the dialyzed extract was digested with 1 mU/mL of heparitinase III (Cat No. H8891; Sigma-Aldrich) at 37°C for 3 hours.

    Techniques: Expressing, Cell Culture, Western Blot

    Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Isolation, Purification, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry, Cytometry, Affinity Purification, Western Blot

    Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Purification, Magnetic Beads, Flow Cytometry, Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

    Model of exosome-target cell interaction mediated by fibronectin. Step 1 , fibronectin ( FN ) is captured on the surface of exosomes by heparan sulfate proteoglycans ( HSPG ). Step 2 , fibronectin, on exosomes binds to heparan sulfate ( HS ) on the surface of the target cell. Step 3 , removal of exosome heparan sulfate or cell surface heparan sulfate with heparitinase, addition of exogenous heparin or heparin mimetics, addition of Hep-II domain-containing fragment of fibronectin, or exposure to antibody against the Hep-II domain of fibronectin dramatically diminishes exosome interaction with target cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Model of exosome-target cell interaction mediated by fibronectin. Step 1 , fibronectin ( FN ) is captured on the surface of exosomes by heparan sulfate proteoglycans ( HSPG ). Step 2 , fibronectin, on exosomes binds to heparan sulfate ( HS ) on the surface of the target cell. Step 3 , removal of exosome heparan sulfate or cell surface heparan sulfate with heparitinase, addition of exogenous heparin or heparin mimetics, addition of Hep-II domain-containing fragment of fibronectin, or exposure to antibody against the Hep-II domain of fibronectin dramatically diminishes exosome interaction with target cells.

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques:

    Exosome-target cell interaction mediated by fibronectin impacts cell behavior. A , exosome interaction with RPMI-8226 cells activates p38 and ERK signaling. An antibody array that simultaneously examines the phosphorylation levels of 43 different protein kinases was utilized to determine what signaling pathways were activated when myeloma-derived exosomes interacted with target cells. RPMI-8226 cells were incubated with or without exosomes isolated from aggressive myeloma cells (CAG cells expressing high heparanase), cell lysates were exposed to membranes, and the membranes were probed with a phosphotyrosine specific antibody. Phosphorylated p38 and ERK ( circled ) were enhanced in cells incubated with the exosomes. The different phospho kinases that are activated in RPMI-8226 cells independent of the addition of exosomes are shown by arrows. Duplicate dots at the corners represent phosphotyrosine positive controls. B , RPMI-8226 cells were treated with exosomes (100 μg/ml) secreted by control or aggressive myeloma cells for 20 min and analyzed for phosphorylated p38 by Western blot. Total p38 serves as the loading control. To determine the role of cell surface heparan sulfate in mediating exosome-induced signaling, RPMI-8266 cells were treated with bacterial heparitinase for 2 h prior to the addition of exosomes from aggressive myeloma cells. C , MMP-9 and DKK1, two downstream target genes of activated p38, are up-regulated following the interaction of myeloma-derived exosomes with the RPMI 8226 cells. RPMI 8266 myeloma cells were incubated with or without exosomes, and DKK1 and MMP-9 mRNA expression in these cells was assessed using real time PCR and normalized using actin expression. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Exosome-target cell interaction mediated by fibronectin impacts cell behavior. A , exosome interaction with RPMI-8226 cells activates p38 and ERK signaling. An antibody array that simultaneously examines the phosphorylation levels of 43 different protein kinases was utilized to determine what signaling pathways were activated when myeloma-derived exosomes interacted with target cells. RPMI-8226 cells were incubated with or without exosomes isolated from aggressive myeloma cells (CAG cells expressing high heparanase), cell lysates were exposed to membranes, and the membranes were probed with a phosphotyrosine specific antibody. Phosphorylated p38 and ERK ( circled ) were enhanced in cells incubated with the exosomes. The different phospho kinases that are activated in RPMI-8226 cells independent of the addition of exosomes are shown by arrows. Duplicate dots at the corners represent phosphotyrosine positive controls. B , RPMI-8226 cells were treated with exosomes (100 μg/ml) secreted by control or aggressive myeloma cells for 20 min and analyzed for phosphorylated p38 by Western blot. Total p38 serves as the loading control. To determine the role of cell surface heparan sulfate in mediating exosome-induced signaling, RPMI-8266 cells were treated with bacterial heparitinase for 2 h prior to the addition of exosomes from aggressive myeloma cells. C , MMP-9 and DKK1, two downstream target genes of activated p38, are up-regulated following the interaction of myeloma-derived exosomes with the RPMI 8226 cells. RPMI 8266 myeloma cells were incubated with or without exosomes, and DKK1 and MMP-9 mRNA expression in these cells was assessed using real time PCR and normalized using actin expression. *, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Ab Array, Derivative Assay, Incubation, Isolation, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Exosome-target cell interaction is mediated by heparan sulfate chains on target cells. A , confocal microscopy analysis of polarized or nonpolarized CAG myeloma cells following addition of CD63-RFP exosomes. Exosomes preferentially interact with heparan sulfate-rich uropods of polarized cells but are widely distributed on nonpolarized cells. B , depletion of heparan sulfate chains on myeloma cells decreases exosome-target cell interaction. RPMI-8226 cells were untreated or treated with heparitinase and washed, and their interactions with exosomes were analyzed by confocal microscopy. Right panel , quantitative data from a similar experiment analyzed by flow cytometry. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Exosome-target cell interaction is mediated by heparan sulfate chains on target cells. A , confocal microscopy analysis of polarized or nonpolarized CAG myeloma cells following addition of CD63-RFP exosomes. Exosomes preferentially interact with heparan sulfate-rich uropods of polarized cells but are widely distributed on nonpolarized cells. B , depletion of heparan sulfate chains on myeloma cells decreases exosome-target cell interaction. RPMI-8226 cells were untreated or treated with heparitinase and washed, and their interactions with exosomes were analyzed by confocal microscopy. Right panel , quantitative data from a similar experiment analyzed by flow cytometry. #, p

    Article Snippet: For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase (Seikagaku) for 2 h at 37 °C followed by extensive washing.

    Techniques: Confocal Microscopy, Flow Cytometry, Cytometry

    Lacritin binding to SDC1 is independent of complete HS/CS glycosaminoglycans.  (A) Lacritin affinity precipitation of human SDC1 multimers stably expressed by HEK293T cells. Lacritin-intein beads were incubated with cell lysates, washed extensively, and treated with heparitinase I/chondroitinase ABC. Pellet (P) and supernatant (S) from the centrifuged digest were then blotted with mAb B-B4 for SDC1 core protein. (B) Lacritin-intein, lacritin-GST, FGF2-GST, intein, and GST beads were incubated with lysates from the same HEK293T cells stably expressing human SDC1. Precipitates were washed, treated, centrifuged, and blotted as in A. (C) Lacritin-intein and FGF2-GST beads were incubated with lysate of HEK293T cells stably expressing human SDC2 or lysate of another HEK293T cell line stably expressing human SDC4. Beads were washed, treated, and centrifuged as in A. Blots were detected with anti-SDC2 mAb L-18 or anti-SDC4 mAb N-19, respectively. A shows both 190- and 80-kD bands. B and C and all subsequent figures show the 80-kD band, which is more predominant in HEK293T transfectants.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin binding to SDC1 is independent of complete HS/CS glycosaminoglycans. (A) Lacritin affinity precipitation of human SDC1 multimers stably expressed by HEK293T cells. Lacritin-intein beads were incubated with cell lysates, washed extensively, and treated with heparitinase I/chondroitinase ABC. Pellet (P) and supernatant (S) from the centrifuged digest were then blotted with mAb B-B4 for SDC1 core protein. (B) Lacritin-intein, lacritin-GST, FGF2-GST, intein, and GST beads were incubated with lysates from the same HEK293T cells stably expressing human SDC1. Precipitates were washed, treated, centrifuged, and blotted as in A. (C) Lacritin-intein and FGF2-GST beads were incubated with lysate of HEK293T cells stably expressing human SDC2 or lysate of another HEK293T cell line stably expressing human SDC4. Beads were washed, treated, and centrifuged as in A. Blots were detected with anti-SDC2 mAb L-18 or anti-SDC4 mAb N-19, respectively. A shows both 190- and 80-kD bands. B and C and all subsequent figures show the 80-kD band, which is more predominant in HEK293T transfectants.

    Article Snippet: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    Techniques: Binding Assay, Affinity Precipitation, Stable Transfection, Incubation, Expressing

    Bacterial heparitinase digestion exposes FGF2-bindable SDC1 to lacritin binding via a domain in SDC1's N-terminal 50 amino acids.  (A) Human SDC1 (lanes 1 and 2), SDC2 (lanes 3 and 4), and SDC4 (lanes 5 and 6) from stably expressing HEK293T cells were individually purified on FGF2-GST, eluted (0.5 and 1 M NaCl), treated with heparitinase I/chondroitinase ABC for 2 h, and incubated with lacritin-intein beads. Blotting is with mAb B-B4 for SDC1, polyclonal antibody L-18 for SDC2, or polyclonal N-19 for SDC4—all core protein specific. (B) Schematic diagram of human SDC1. The dotted line indicates truncation sites in the ectodomain forming the deletion constructs del 1–51, 51–252, and 51–310. Boxes represent PSIPRED-predicted α helices. Wavy lines represent HS and CS. TM, transmembrane domain. (C) Comparative incubation of FGF2-GST and lacritin-intein beads with human SDC1 or human SDC1 del 1–51 lysates from stably expressing HEK293T cells. After incubation, beads were washed extensively and either treated with heparitinase I/chondroitinase ABC (+) or left untreated (−). Beads were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Lysate from HEK293T cells stably expressing SDC1 del 1–51 is blotted in lanes 6 and 7. (D) Comparative incubation of lacritin-intein beads with human SDC1 del 51–252, 1–51, or 51–310 lysates from stably or transiently expressing HEK293T cells. pcDNA is lysate from cells transfected with vector only. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. Beads were centrifuged, and pellets were blotted with mAb 3G10 for desaturated uronates in SDC1.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Bacterial heparitinase digestion exposes FGF2-bindable SDC1 to lacritin binding via a domain in SDC1's N-terminal 50 amino acids. (A) Human SDC1 (lanes 1 and 2), SDC2 (lanes 3 and 4), and SDC4 (lanes 5 and 6) from stably expressing HEK293T cells were individually purified on FGF2-GST, eluted (0.5 and 1 M NaCl), treated with heparitinase I/chondroitinase ABC for 2 h, and incubated with lacritin-intein beads. Blotting is with mAb B-B4 for SDC1, polyclonal antibody L-18 for SDC2, or polyclonal N-19 for SDC4—all core protein specific. (B) Schematic diagram of human SDC1. The dotted line indicates truncation sites in the ectodomain forming the deletion constructs del 1–51, 51–252, and 51–310. Boxes represent PSIPRED-predicted α helices. Wavy lines represent HS and CS. TM, transmembrane domain. (C) Comparative incubation of FGF2-GST and lacritin-intein beads with human SDC1 or human SDC1 del 1–51 lysates from stably expressing HEK293T cells. After incubation, beads were washed extensively and either treated with heparitinase I/chondroitinase ABC (+) or left untreated (−). Beads were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Lysate from HEK293T cells stably expressing SDC1 del 1–51 is blotted in lanes 6 and 7. (D) Comparative incubation of lacritin-intein beads with human SDC1 del 51–252, 1–51, or 51–310 lysates from stably or transiently expressing HEK293T cells. pcDNA is lysate from cells transfected with vector only. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. Beads were centrifuged, and pellets were blotted with mAb 3G10 for desaturated uronates in SDC1.

    Article Snippet: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    Techniques: Binding Assay, Stable Transfection, Expressing, Purification, Incubation, Construct, Transfection, Plasmid Preparation

    Lacritin's C terminus binds SDC1.  (A) Schematic diagram of lacritin with dotted lines indicating N- and C-terminal truncations. All lacritin truncations were expressed as intein fusion proteins for affinity precipitation. The asterisk indicates mitogenic domain (  Wang et al., 2006 ), and boxes represent PSIPRED-predicted α helices. (B) Lacritin-, C-5–, C-10–, C-15–, C-25–, and C-59–intein beads were incubated with lysates from HEK293T cells stably expressing human SDC1. Beads were washed and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) blotted with mAb B-B4 for SDC1 core protein, all as in   Fig. 2 . (C) Incubation of lacritin-, N-15– and N-24–intein beads with the same human SDC1 lysates was followed with identical washing, heparitinase I/chondroitinase ABC digestion, centrifugation, and B-B4 mAb blotting. Lys, lysate.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin's C terminus binds SDC1. (A) Schematic diagram of lacritin with dotted lines indicating N- and C-terminal truncations. All lacritin truncations were expressed as intein fusion proteins for affinity precipitation. The asterisk indicates mitogenic domain ( Wang et al., 2006 ), and boxes represent PSIPRED-predicted α helices. (B) Lacritin-, C-5–, C-10–, C-15–, C-25–, and C-59–intein beads were incubated with lysates from HEK293T cells stably expressing human SDC1. Beads were washed and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) blotted with mAb B-B4 for SDC1 core protein, all as in Fig. 2 . (C) Incubation of lacritin-, N-15– and N-24–intein beads with the same human SDC1 lysates was followed with identical washing, heparitinase I/chondroitinase ABC digestion, centrifugation, and B-B4 mAb blotting. Lys, lysate.

    Article Snippet: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    Techniques: Affinity Precipitation, Incubation, Stable Transfection, Expressing, Centrifugation

    Lacritin-SDC1 binding is inhibited by soluble hS1ED, lacritin, and N-24, but not by C-25, C-59, HS, CS, SDC2, or SDC4.  (A, top) Lacritin-intein beads were incubated with human SDC1 lysates from stably expressing HEK293T cells in the presence of increasing amounts of soluble HS (70–700 μg), HS (700 μg) plus CS (700 μg), or lacritin (14–700 μg) or no inhibitor (−). The quantity of soluble inhibitor was calibrated relative to the ∼7–8 μg of human SDC1 elutable from lacritin-intein beads with 1 M NaCl. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets were blotted with mAb B-B4 for SDC1 core protein, as in   Fig. 2 . (bottom) Lacritin-intein beads were incubated with human SDC1 lysates in the presence of soluble N-24, C-25, C-59 (14 μg of each), increasing amounts of bacterially expressed human SDC1 ectodomain (hS1ED; 35–700 μg), or with HEK293T cell–expressed native SDC2 or -4 (70 μg of each). Beads were washed and treated as above. (B) Quantification of inhibition binding. Error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin-SDC1 binding is inhibited by soluble hS1ED, lacritin, and N-24, but not by C-25, C-59, HS, CS, SDC2, or SDC4. (A, top) Lacritin-intein beads were incubated with human SDC1 lysates from stably expressing HEK293T cells in the presence of increasing amounts of soluble HS (70–700 μg), HS (700 μg) plus CS (700 μg), or lacritin (14–700 μg) or no inhibitor (−). The quantity of soluble inhibitor was calibrated relative to the ∼7–8 μg of human SDC1 elutable from lacritin-intein beads with 1 M NaCl. After incubation, beads were washed extensively and treated with heparitinase I/chondroitinase ABC. The digests were centrifuged, and pellets were blotted with mAb B-B4 for SDC1 core protein, as in Fig. 2 . (bottom) Lacritin-intein beads were incubated with human SDC1 lysates in the presence of soluble N-24, C-25, C-59 (14 μg of each), increasing amounts of bacterially expressed human SDC1 ectodomain (hS1ED; 35–700 μg), or with HEK293T cell–expressed native SDC2 or -4 (70 μg of each). Beads were washed and treated as above. (B) Quantification of inhibition binding. Error bars indicate SEM.

    Article Snippet: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    Techniques: Binding Assay, Incubation, Stable Transfection, Expressing, Inhibition

    Lacritin and FGF2 bind different forms of cell surface SDC1.  (A) Sequential affinity precipitation assays. Lanes 1–3 show lysate from human SDC1 stably expressing HEK293T cells sequentially incubated with three rounds of fresh FGF2-GST beads. Half of the final depleted lysate was then incubated with lacritin-intein beads (lane 4), and the other half was methanol precipitated (lane 9). Similarly, in lanes 5–7, a different aliquot of lysate from the same cells was sequentially incubated with three rounds of fresh lacritin-intein beads. Half of the final depleted lysate was then incubated with FGF2-GST beads (lane 8), and the other half was methanol precipitated (lane 10). Beads were washed and treated with heparitinase I/ chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Shown are digest supernatants (lanes 1–3 and 8) and pellets (lanes 4–7) as per heparitinase release of FGF2-bound or resistance of lacritin-bound SDC1, respectively. (B) HEK293T cells stably expressing human SDC1 were either lysed as usual or first briefly trypsinized (

    Journal: The Journal of Cell Biology

    Article Title: Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

    doi: 10.1083/jcb.200511134

    Figure Lengend Snippet: Lacritin and FGF2 bind different forms of cell surface SDC1. (A) Sequential affinity precipitation assays. Lanes 1–3 show lysate from human SDC1 stably expressing HEK293T cells sequentially incubated with three rounds of fresh FGF2-GST beads. Half of the final depleted lysate was then incubated with lacritin-intein beads (lane 4), and the other half was methanol precipitated (lane 9). Similarly, in lanes 5–7, a different aliquot of lysate from the same cells was sequentially incubated with three rounds of fresh lacritin-intein beads. Half of the final depleted lysate was then incubated with FGF2-GST beads (lane 8), and the other half was methanol precipitated (lane 10). Beads were washed and treated with heparitinase I/ chondroitinase ABC. The digests were centrifuged, and pellets (P) and supernatants (S) were blotted with mAb B-B4 for SDC1 core protein. Shown are digest supernatants (lanes 1–3 and 8) and pellets (lanes 4–7) as per heparitinase release of FGF2-bound or resistance of lacritin-bound SDC1, respectively. (B) HEK293T cells stably expressing human SDC1 were either lysed as usual or first briefly trypsinized (

    Article Snippet: For SDS-PAGE and immunoblotting, beads were digested with heparitinase I (Seikagaku America) and chondroitin ABC lyase (MP Biochemicals) because native syndecans migrate as a heterodisperse smear in SDS-PAGE.

    Techniques: Affinity Precipitation, Stable Transfection, Expressing, Incubation

    Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.

    Journal: The Journal of Cell Biology

    Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

    doi:

    Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.

    Article Snippet: Heparinase I ( Sigma Chemical Co. ) was used at 600 mU, and heparitinase II (Seikagaku Corporation, Tokyo, Japan) was used at 4 mU.

    Techniques: Immunofluorescence

    Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated BAEC with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/μ were as follows: control, 86%; heparinase I, 54%; heparitinase II, 56%; chondroitinase, 89%; hyaluronidase, 90%; trypsin, 7%. The background staining with CD4/μ chimera was

    Journal: The Journal of Cell Biology

    Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

    doi:

    Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated BAEC with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/μ were as follows: control, 86%; heparinase I, 54%; heparitinase II, 56%; chondroitinase, 89%; hyaluronidase, 90%; trypsin, 7%. The background staining with CD4/μ chimera was

    Article Snippet: Heparinase I ( Sigma Chemical Co. ) was used at 600 mU, and heparitinase II (Seikagaku Corporation, Tokyo, Japan) was used at 4 mU.

    Techniques: Immunofluorescence, Staining

    HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.

    Journal: BMC Developmental Biology

    Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

    doi: 10.1186/1471-213X-11-38

    Figure Lengend Snippet: HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.

    Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 4.2.2.8) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.

    Techniques: Negative Control, Incubation

    Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.

    Journal: BMC Developmental Biology

    Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

    doi: 10.1186/1471-213X-11-38

    Figure Lengend Snippet: Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.

    Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 4.2.2.8) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.

    Techniques: Staining, Immunohistochemistry

    HS 'phage display antibodies identify distinct epitopes in situ . In fetal rat lungs, HS antibodies display different patterns of staining. HS3B7V exclusively labels epithelial basement membranes, whereas HS4E4V and HS3A8V show a more widespread staining pattern. In addition to epithelial basement membrane staining, HS4E4V labels sub-epithelial mesenchymal cells surrounding smaller distal airways and HS3A8V highlights the entire lung mesenchyme and in addition, stains epithelial cells at E15.5. One antibody, HS4C3V, did not stain fetal rat lungs of any developmental age; however, positive staining of adult rat kidney confirmed the functionality of HS4C3V in immunohistochemistry. E15.5 and E17.5 rat lungs and adult rat kidney were probed with HS antibodies followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Negative controls were omission of HS antibody or digestion of HS with heparitinase prior to antibody incubation (HS4E4V shown, heparitinase digest controls for other antibodies are shown in additional files). Scale bar represents 10 μm and all images are the same magnification. (ep) epithelium, (me) mesenchyme, (bm) basement membrane, (aw) airway, (G) glomerulus, (cap) peritubular capillary.

    Journal: BMC Developmental Biology

    Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

    doi: 10.1186/1471-213X-11-38

    Figure Lengend Snippet: HS 'phage display antibodies identify distinct epitopes in situ . In fetal rat lungs, HS antibodies display different patterns of staining. HS3B7V exclusively labels epithelial basement membranes, whereas HS4E4V and HS3A8V show a more widespread staining pattern. In addition to epithelial basement membrane staining, HS4E4V labels sub-epithelial mesenchymal cells surrounding smaller distal airways and HS3A8V highlights the entire lung mesenchyme and in addition, stains epithelial cells at E15.5. One antibody, HS4C3V, did not stain fetal rat lungs of any developmental age; however, positive staining of adult rat kidney confirmed the functionality of HS4C3V in immunohistochemistry. E15.5 and E17.5 rat lungs and adult rat kidney were probed with HS antibodies followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Negative controls were omission of HS antibody or digestion of HS with heparitinase prior to antibody incubation (HS4E4V shown, heparitinase digest controls for other antibodies are shown in additional files). Scale bar represents 10 μm and all images are the same magnification. (ep) epithelium, (me) mesenchyme, (bm) basement membrane, (aw) airway, (G) glomerulus, (cap) peritubular capillary.

    Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 4.2.2.8) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.

    Techniques: In Situ, Staining, Immunohistochemistry, Incubation

    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

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Journal: Scientific Reports

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    doi: 10.1038/s41598-018-27566-z

    Figure Lengend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Article Snippet: Heparinase III (R & D Systems) was used at 40 micrograms per milliliter.

    Techniques:

    Western blot of total L2 proteoglycans probed with affinity-purified R664 polyclonal antibodies against a fusion protein prepared using clone F15a (see Fig. 1 ). The proteoglycans were untreated (lane 1 ) or pretreated with heparinase III (lanes 2 and 3 ) and/or chondroitinase ABC (lanes 3 and 4 ). A core protein is only clearly resolved after removal of chondroitin sulfate chains. The migration of molecular mass markers in kD is shown.

    Journal: The Journal of Cell Biology

    Article Title: cDNA Cloning of the Basement Membrane Chondroitin Sulfate Proteoglycan Core Protein, Bamacan: A Five Domain Structure Including Coiled-Coil Motifs

    doi:

    Figure Lengend Snippet: Western blot of total L2 proteoglycans probed with affinity-purified R664 polyclonal antibodies against a fusion protein prepared using clone F15a (see Fig. 1 ). The proteoglycans were untreated (lane 1 ) or pretreated with heparinase III (lanes 2 and 3 ) and/or chondroitinase ABC (lanes 3 and 4 ). A core protein is only clearly resolved after removal of chondroitin sulfate chains. The migration of molecular mass markers in kD is shown.

    Article Snippet: These were digested with 10 mU chondroitinase ABC (EC 4.2.2.4) or 20 mU heparinase III (EC 4.2.2.8; Seikagaku America, Ijamsville, MD) for 3 h or overnight at 37°C, respectively.

    Techniques: Western Blot, Affinity Purification, Migration

    Glycanation of the NH 2 -terminal region of bamacan domain V. Autoradiogram of 5–15% gradient SDS-PAGE of protein A–bamacan fusion protein, heparinase III treated (lane 1 ), chondroitinase ABC treated (lane 2 ), or untreated (lane 5 ). Only after chondroitinase ABC treatment is a discrete protein resolved ( arrows denote fusion protein of M r ∼ 50K). (Lane 3 ) Result from transfection of empty pRK5F10 vector; (lane 4 ) equivalent from empty pcDNA 3 vector (which fails to bind Ig-agarose).

    Journal: The Journal of Cell Biology

    Article Title: cDNA Cloning of the Basement Membrane Chondroitin Sulfate Proteoglycan Core Protein, Bamacan: A Five Domain Structure Including Coiled-Coil Motifs

    doi:

    Figure Lengend Snippet: Glycanation of the NH 2 -terminal region of bamacan domain V. Autoradiogram of 5–15% gradient SDS-PAGE of protein A–bamacan fusion protein, heparinase III treated (lane 1 ), chondroitinase ABC treated (lane 2 ), or untreated (lane 5 ). Only after chondroitinase ABC treatment is a discrete protein resolved ( arrows denote fusion protein of M r ∼ 50K). (Lane 3 ) Result from transfection of empty pRK5F10 vector; (lane 4 ) equivalent from empty pcDNA 3 vector (which fails to bind Ig-agarose).

    Article Snippet: These were digested with 10 mU chondroitinase ABC (EC 4.2.2.4) or 20 mU heparinase III (EC 4.2.2.8; Seikagaku America, Ijamsville, MD) for 3 h or overnight at 37°C, respectively.

    Techniques: SDS Page, Transfection, Plasmid Preparation

    GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or Heparinase III treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .

    Journal: Neuron

    Article Title: An input-specific orphan receptor GPR158-HSPG interaction organizes hippocampal mossy fiber-CA3 synapses

    doi: 10.1016/j.neuron.2018.08.038

    Figure Lengend Snippet: GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or Heparinase III treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .

    Article Snippet: For the analysis of heparinase III treatment, hippocampal neurons (7 DIV) were treated with 1 U/ml heparinase III (Sigma-Aldrich) or vehicle (20 mM Tris-HCl [pH 7.5], 0.1 mg/ml BSA, 4mM CaCl2 ) for 2 hours at 37°C.

    Techniques: Binding Assay, Mass Spectrometry, Affinity Purification, Mutagenesis, Expressing, Transfection, Cell Culture, Negative Control

    The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of heparitinase treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.

    Journal: The American Journal of Pathology

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

    doi:

    Figure Lengend Snippet: The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of heparitinase treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody ( top ). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody ( bottom ). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.

    Article Snippet: For enzymatic cleavage of GAGs, cells were treated with either heparitinase ( Flafobacterium heparinum , EC 4.2.2.8; ICN Biomedicals, Aurora, OH) or chondroitinase avidin-biotin-peroxidase complex ( Proteus vulgaris , EC 4.2.2.4; Boehringer Mannheim, Almere, The Netherlands) in phosphate-buffered saline (PBS) at 37°C for the periods indicated.

    Techniques: Immunoprecipitation, Activation Assay, Binding Assay, Mutagenesis

    CD44v3 isoforms on colon carcinoma cell lines are decorated with HS. CD44v3 was immunoprecipitated from the colon carcinoma cell lines SW480 and HT-29, and, as a positive control, from Namalwa cells transfected with CD44v3–10, with mouse anti-CD44v3. Before immunoprecipitation, the cells were treated with either PBS (−), 30 mU/ml heparitinase (HT), or 30 mU/ml chondroitinase ABC (CH) at 37°C for 3.5 hours. The Western blot of the precipitates was stained with the anti-pan CD44 mAb Hermes-3, stripped, and restained with the mAb 3G10 that detects ΔHS stubs after treatment of HS with heparitinase. CD44v3 isoforms decorated with HS are indicated with arrows .

    Journal: The American Journal of Pathology

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

    doi:

    Figure Lengend Snippet: CD44v3 isoforms on colon carcinoma cell lines are decorated with HS. CD44v3 was immunoprecipitated from the colon carcinoma cell lines SW480 and HT-29, and, as a positive control, from Namalwa cells transfected with CD44v3–10, with mouse anti-CD44v3. Before immunoprecipitation, the cells were treated with either PBS (−), 30 mU/ml heparitinase (HT), or 30 mU/ml chondroitinase ABC (CH) at 37°C for 3.5 hours. The Western blot of the precipitates was stained with the anti-pan CD44 mAb Hermes-3, stripped, and restained with the mAb 3G10 that detects ΔHS stubs after treatment of HS with heparitinase. CD44v3 isoforms decorated with HS are indicated with arrows .

    Article Snippet: For enzymatic cleavage of GAGs, cells were treated with either heparitinase ( Flafobacterium heparinum , EC 4.2.2.8; ICN Biomedicals, Aurora, OH) or chondroitinase avidin-biotin-peroxidase complex ( Proteus vulgaris , EC 4.2.2.4; Boehringer Mannheim, Almere, The Netherlands) in phosphate-buffered saline (PBS) at 37°C for the periods indicated.

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

    En face and cross sections of RFPEC in which HS was immunostained in green and cell nuclei in blue. Notes: ( A ) Control, ( B ) low serum, ( C ) Hep III, and ( D ) Hep III + HS. ( E ) Thickness measurements of the glycocalyx when observing only the HS component by binding the 10E4 epitope HS antibody to the glycosaminoglycan under control, low serum, Hep III, and Hep III + HS treatments. HS thickness decreased significantly for all treatments. ( F ) HS coverage of RFPEC after various treatments. HS coverage decreased significantly after heparinase III treatment and recovered after HS addition. * P

    Journal: International Journal of Nanomedicine

    Article Title: Endothelial glycocalyx conditions influence nanoparticle uptake for passive targeting

    doi: 10.2147/IJN.S106299

    Figure Lengend Snippet: En face and cross sections of RFPEC in which HS was immunostained in green and cell nuclei in blue. Notes: ( A ) Control, ( B ) low serum, ( C ) Hep III, and ( D ) Hep III + HS. ( E ) Thickness measurements of the glycocalyx when observing only the HS component by binding the 10E4 epitope HS antibody to the glycosaminoglycan under control, low serum, Hep III, and Hep III + HS treatments. HS thickness decreased significantly for all treatments. ( F ) HS coverage of RFPEC after various treatments. HS coverage decreased significantly after heparinase III treatment and recovered after HS addition. * P

    Article Snippet: For the degraded glycocalyx configuration, cultures were pretreated with DMEM/10% FBS/1% penicillin-streptomycin containing a 2.5×10−6 IU heparinase III (Hep III) (IBEX Pharmaceutical, Montreal, QC, Canada) enzyme for 2 hours, selectively degrading the HS glycosaminoglycans of the glycocalyx.

    Techniques: Binding Assay

    Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by heparinase treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from three independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins

    doi: 10.1101/2020.08.18.255810

    Figure Lengend Snippet: Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by heparinase treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from three independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.

    Article Snippet: Biosynthesis inhibition and enzymatic treatmentHuH7.5 cells were treated in D-PBS/0.25% BSA with 46 miliunits heparinase III (Amsbio) for 1 hour at 37°C, washed and used in subsequent experiments.

    Techniques: Binding Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Luciferase, Two Tailed Test

    ( A ) ACE2 cell surface expression on Namatwa cell line, DCs, LCs and Huh 7.5. Representative data for an experiment repeated more than three times with similar results. ( B ) Huh7.5 were left untreated or treated with heparinase for 1 hand heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. One representative donor out of 3 is depicted. ( C ) Cell surface expression of ACE2 on 293T and Caco2 cell lines was determined by real-time PCR. ( D ) LC were stained with antibodies against CD207 and CD1 a and analysed by flow cytometry. The histogram shows the cell surface expression of the receptor. Representative data for an experiment repeated more than three times with similar results. Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVA with Tukey’s multiple-comparison test. ****p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins

    doi: 10.1101/2020.08.18.255810

    Figure Lengend Snippet: ( A ) ACE2 cell surface expression on Namatwa cell line, DCs, LCs and Huh 7.5. Representative data for an experiment repeated more than three times with similar results. ( B ) Huh7.5 were left untreated or treated with heparinase for 1 hand heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. One representative donor out of 3 is depicted. ( C ) Cell surface expression of ACE2 on 293T and Caco2 cell lines was determined by real-time PCR. ( D ) LC were stained with antibodies against CD207 and CD1 a and analysed by flow cytometry. The histogram shows the cell surface expression of the receptor. Representative data for an experiment repeated more than three times with similar results. Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVA with Tukey’s multiple-comparison test. ****p

    Article Snippet: Biosynthesis inhibition and enzymatic treatmentHuH7.5 cells were treated in D-PBS/0.25% BSA with 46 miliunits heparinase III (Amsbio) for 1 hour at 37°C, washed and used in subsequent experiments.

    Techniques: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining