heparinase Search Results


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  • 94
    New England Biolabs bacteroides heparinase i
    Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides <t>Heparinase</t> I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.
    Bacteroides Heparinase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore heparinase i
    LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither <t>heparinase</t> ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.
    Heparinase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 886 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 776 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore heparinase ii
    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 Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore flavobacterium heparinum
    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase <t>I/III</t> blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P
    Flavobacterium Heparinum, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku heparinase
    N-sulfate-enriched microenvironment forms a belt around the demyelinated lesion. HS (10E4) labeling on the contra- ( A ) and ipsi- ( B–C ) lateral side to the lesion illustrates the generation of a N-sulfated microenvironment surrounding the lesion (delimited by white dashed lines) at five dpi (n = 3). No immunoreactivity was found after <t>Heparinase</t> I treatment ( D ) thus validating the 10E4 antibody specificity. Scale bars: 20 µm in A, B, D; 10 µm in C. CC, corpus callosum; V, ventricle.
    Heparinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    IBEX Technologies heparinase iii
    Effects of PD98059 and <t>heparinase</t> on SMC marker gene expression under 3-D interstitial flow. PD98059 and heparinase reverse interstitial flow-induced reductions in SM-MHC, smoothelin, and calponin expression, but further enhance α-SMA and SM22 expression in 3-D. SMCs (A) and MFBs (B) in collagen gels were pretreated with PD98059 (PD) or heparinase <t>III</t> (Hepr) for 3 h, and then exposed to interstitial flow (1 cmH 2 O) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P
    Heparinase Iii, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Seikagaku heparinase iii
    GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K d s were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125 I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with <t>heparinase</t> <t>III</t> (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125 I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.
    Heparinase Iii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    IBEX Technologies heparinase i
    Both HS and protein components of SCM are required for boundary formation Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); <t>heparinase</t> treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75 NTR (green). The number of cells mingling with astrocytes was counted across a 300 μm line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate ± SEM. Scale bar 50 μm. ** p
    Heparinase I, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 92/100, based on 35 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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    88
    Seikagaku heparin lyase iii
    Posterior mouse extracellular matrix <t>(ECM)</t> induces pattern formation by axolotl blastema cells. (A) Schematic diagram of surgical procedures. Mouse hindlimb (femur) skin samples (red) were collected from mice for ECM extraction followed by grafting into an axolotl skin wound with a deviated brachial nerve (blue). (B) Differential regenerative responses to grafts of mouse‐derived anterior or posterior ECM from skin samples at postnatal day 3−4 either with or without heparin lyase <t>III</t> treatment. Ant, anterior; Post, posterior; (−)HS, heparin lyase III treatment to remove heparan sulfate from the ECM. (C) Whole‐mount skeletal preparation of a skeletal‐like element formed by axolotl blastema cells in response to an ECM graft from the posterior of a mouse limb at about 2 months post‐grafting: red, ossified bone; blue, cartilage indicated by arrow.
    Heparin Lyase Iii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AMS Biotechnology heparinase iii
    Posterior mouse extracellular matrix <t>(ECM)</t> induces pattern formation by axolotl blastema cells. (A) Schematic diagram of surgical procedures. Mouse hindlimb (femur) skin samples (red) were collected from mice for ECM extraction followed by grafting into an axolotl skin wound with a deviated brachial nerve (blue). (B) Differential regenerative responses to grafts of mouse‐derived anterior or posterior ECM from skin samples at postnatal day 3−4 either with or without heparin lyase <t>III</t> treatment. Ant, anterior; Post, posterior; (−)HS, heparin lyase III treatment to remove heparan sulfate from the ECM. (C) Whole‐mount skeletal preparation of a skeletal‐like element formed by axolotl blastema cells in response to an ECM graft from the posterior of a mouse limb at about 2 months post‐grafting: red, ossified bone; blue, cartilage indicated by arrow.
    Heparinase Iii, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs bacteroides heparinase ii
    Posterior mouse extracellular matrix <t>(ECM)</t> induces pattern formation by axolotl blastema cells. (A) Schematic diagram of surgical procedures. Mouse hindlimb (femur) skin samples (red) were collected from mice for ECM extraction followed by grafting into an axolotl skin wound with a deviated brachial nerve (blue). (B) Differential regenerative responses to grafts of mouse‐derived anterior or posterior ECM from skin samples at postnatal day 3−4 either with or without heparin lyase <t>III</t> treatment. Ant, anterior; Post, posterior; (−)HS, heparin lyase III treatment to remove heparan sulfate from the ECM. (C) Whole‐mount skeletal preparation of a skeletal‐like element formed by axolotl blastema cells in response to an ECM graft from the posterior of a mouse limb at about 2 months post‐grafting: red, ossified bone; blue, cartilage indicated by arrow.
    Bacteroides Heparinase Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Journal: Scientific Reports

    Article Title: Heparin affinity purification of extracellular vesicles

    doi: 10.1038/srep10266

    Figure Lengend Snippet: Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Article Snippet: Beads were then washed three times with PBS 1x and either treated with Bacteroides Heparinase I (New England Biolabs, Ipswich, MA; 60U/ml heparin beads) according to the manufacturer’s recommendations or mock treated (incubation buffer devoid of heparinase) for 1 hr at 30 °C.

    Techniques: Isolation, Purification, Incubation, Biomarker Assay, Derivative Assay

    LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.

    Journal: Glycobiology

    Article Title: LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

    doi: 10.1093/glycob/cww075

    Figure Lengend Snippet: LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.

    Article Snippet: Heparinase treatment and HF dephosphorylation For heparinase treatment, samples were treated with a mixture of heparinases I and III (Sigma, 0.6 and 0.3 Sigma units, respectively) in 0.

    Techniques: Binding Assay, Construct, Sequencing, Purification, Expressing, Functional Assay, Modification

    Characterization of SDC 4–promyostatin interaction. (A) Co‐immunoprecipitations (co‐ IP s) were carried out with rabbit antiserum to the C‐terminal part of myostatin ( AB 3239) in un‐injured (control, day 0, d0) and injured (3 days after notexin injury, d3) soleus muscle homogenates. Different volumes (20 and 7 μL) of the eluted immunocomplex were loaded in case of the injured sample. The blots were reacted with antibodies to SDC 4 raised in goat. Myostatin co‐immunoprecipitated SDC 4 in both cases. Input lanes represent the total homogenates; 10% of the total protein amount used in co‐ IP was loaded. (B) Co‐ IP assays were performed with anti‐ SDC 4 antibodies, and the blots were reacted with anti‐myostatin antisera ( AB 3239). The negative control was incubated only with the secondary antibody. For the input lanes, 10% of the total protein amount used in co‐ IP was loaded. The additional band at ~ 42 kD a in d0 input can be a processing intermediate of promyostatin. (C) Heparan sulfate chains were digested in injured samples (d3; sample 1 and 2) with heparinase II enzyme following immunoprecipitation with goat anti‐ SDC 4 antibody. Different amounts (25 and 5 μL) of the eluted volume of the heparinase II digested immunoprecipitate of sample 1 were loaded. We could not detect promyostatin in the immunoprecipitate after heparinase digestion. For the input lanes, 7.5% of the total protein amount used in co‐ IP s were loaded. Note, that both promyostatin and mature myostatin were detected in the supernatant (digestion buffer) after heparinase digestion.

    Journal: Febs Letters

    Article Title: Syndecan‐4 influences mammalian myoblast proliferation by modulating myostatin signalling and G1/S transition

    doi: 10.1002/1873-3468.13227

    Figure Lengend Snippet: Characterization of SDC 4–promyostatin interaction. (A) Co‐immunoprecipitations (co‐ IP s) were carried out with rabbit antiserum to the C‐terminal part of myostatin ( AB 3239) in un‐injured (control, day 0, d0) and injured (3 days after notexin injury, d3) soleus muscle homogenates. Different volumes (20 and 7 μL) of the eluted immunocomplex were loaded in case of the injured sample. The blots were reacted with antibodies to SDC 4 raised in goat. Myostatin co‐immunoprecipitated SDC 4 in both cases. Input lanes represent the total homogenates; 10% of the total protein amount used in co‐ IP was loaded. (B) Co‐ IP assays were performed with anti‐ SDC 4 antibodies, and the blots were reacted with anti‐myostatin antisera ( AB 3239). The negative control was incubated only with the secondary antibody. For the input lanes, 10% of the total protein amount used in co‐ IP was loaded. The additional band at ~ 42 kD a in d0 input can be a processing intermediate of promyostatin. (C) Heparan sulfate chains were digested in injured samples (d3; sample 1 and 2) with heparinase II enzyme following immunoprecipitation with goat anti‐ SDC 4 antibody. Different amounts (25 and 5 μL) of the eluted volume of the heparinase II digested immunoprecipitate of sample 1 were loaded. We could not detect promyostatin in the immunoprecipitate after heparinase digestion. For the input lanes, 7.5% of the total protein amount used in co‐ IP s were loaded. Note, that both promyostatin and mature myostatin were detected in the supernatant (digestion buffer) after heparinase digestion.

    Article Snippet: The immunoprecipitate was resuspended in heparinase buffer and digested with 0.4 mU heparinase II enzyme (Sigma‐Aldrich) for 3 h at 37 °C.

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Incubation

    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

    Double-labeled ( closed circles , [ 3 H]glucosamine; open circles , [ 35 S]sulfate) β-TC3 HS following application to and elution from an IAPP affinity column, and following heparinase I or heparinase III digestion. Reaction products were separated by Bio-gel P-10 size exclusion chromatography. IAPP-bound HS is shown in A (heparinase I digested) and B (heparinase III digested), whereas non-bound HS is shown in C (heparinase I digested) and D (heparinase III digested). β-TC3 HS showed similar composition regardless of IAPP binding ability, with both bound and non-bound material being relatively insensitive to heparinase I treatment but sensitive to heparinase III treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: Overall Sulfation of Heparan Sulfate from Pancreatic Islet ?-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide *

    doi: 10.1074/jbc.M112.409847

    Figure Lengend Snippet: Double-labeled ( closed circles , [ 3 H]glucosamine; open circles , [ 35 S]sulfate) β-TC3 HS following application to and elution from an IAPP affinity column, and following heparinase I or heparinase III digestion. Reaction products were separated by Bio-gel P-10 size exclusion chromatography. IAPP-bound HS is shown in A (heparinase I digested) and B (heparinase III digested), whereas non-bound HS is shown in C (heparinase I digested) and D (heparinase III digested). β-TC3 HS showed similar composition regardless of IAPP binding ability, with both bound and non-bound material being relatively insensitive to heparinase I treatment but sensitive to heparinase III treatment.

    Article Snippet: Heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycans were selectively removed from the immobilized proteoglycans by heparinase digestion (2.6 units/ml heparinase I, 1.3 units/ml heparinase II + 2.6 units/ml heparinase III, Sigma) or chondroitin ABC lyase digestion (0.1 units/ml), respectively, for 2 h at 37 °C.

    Techniques: Labeling, Affinity Column, Size-exclusion Chromatography, Binding Assay

    Double-labeled ( closed symbols , [ 3 H]glucosamine; open symbols , [ 35 S]sulfate) HS from β-TC3 cells ( A ) and NMuMG cells ( B ) was subjected to Sepharose CL-6B size exclusion chromatography prior to ( circles ) or following heparinase I digestion ( triangles ), which cleaves highly sulfated regions of heparan sulfate. Intact β-TC3 heparan sulfate eluted at K av = 0.6, whereas two HS peaks of approximately equal abundance were observed after heparinase I treatment: at K av = 0.58 and 0.8. Thus, β-TC3 HS was only partially sensitive to heparinase I digestion, demonstrating poor overall sulfation. Intact NMuMG HS eluted at K av = 0.45, whereas following heparinase I digestion, a larger peak was present at K av = 0.7 and a smaller peak at K av = 0.9. Thus, NMuMG HS was more sensitive to heparinase I digestion, demonstrating higher overall sulfation in this HS preparation.

    Journal: The Journal of Biological Chemistry

    Article Title: Overall Sulfation of Heparan Sulfate from Pancreatic Islet ?-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide *

    doi: 10.1074/jbc.M112.409847

    Figure Lengend Snippet: Double-labeled ( closed symbols , [ 3 H]glucosamine; open symbols , [ 35 S]sulfate) HS from β-TC3 cells ( A ) and NMuMG cells ( B ) was subjected to Sepharose CL-6B size exclusion chromatography prior to ( circles ) or following heparinase I digestion ( triangles ), which cleaves highly sulfated regions of heparan sulfate. Intact β-TC3 heparan sulfate eluted at K av = 0.6, whereas two HS peaks of approximately equal abundance were observed after heparinase I treatment: at K av = 0.58 and 0.8. Thus, β-TC3 HS was only partially sensitive to heparinase I digestion, demonstrating poor overall sulfation. Intact NMuMG HS eluted at K av = 0.45, whereas following heparinase I digestion, a larger peak was present at K av = 0.7 and a smaller peak at K av = 0.9. Thus, NMuMG HS was more sensitive to heparinase I digestion, demonstrating higher overall sulfation in this HS preparation.

    Article Snippet: Heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycans were selectively removed from the immobilized proteoglycans by heparinase digestion (2.6 units/ml heparinase I, 1.3 units/ml heparinase II + 2.6 units/ml heparinase III, Sigma) or chondroitin ABC lyase digestion (0.1 units/ml), respectively, for 2 h at 37 °C.

    Techniques: Labeling, Size-exclusion Chromatography

    HS plays a role in henipavirus infection. (A) Vero cells were either treated with heparinase 3 or left untreated prior to NiV infection. Titration was performed 3 days later in a plaque assay. (B and C) Vero cells were treated with increasing concentrations of sodium chlorate for 48 h and then infected with either NiV (B) or HeV (C); titration was performed 3 days later in a plaque assay. Results are expressed as a percentage of results for nontreated controls from triplicate cultures, ± the SD. *, P

    Journal: mBio

    Article Title: Heparan Sulfate-Dependent Enhancement of Henipavirus Infection

    doi: 10.1128/mBio.02427-14

    Figure Lengend Snippet: HS plays a role in henipavirus infection. (A) Vero cells were either treated with heparinase 3 or left untreated prior to NiV infection. Titration was performed 3 days later in a plaque assay. (B and C) Vero cells were treated with increasing concentrations of sodium chlorate for 48 h and then infected with either NiV (B) or HeV (C); titration was performed 3 days later in a plaque assay. Results are expressed as a percentage of results for nontreated controls from triplicate cultures, ± the SD. *, P

    Article Snippet: In some experiments, cells were incubated for 1 h at 37°C with 10 U/ml of heparinase 3 (Sigma) and washed 3 times before contact with NiV.

    Techniques: Infection, Titration, Plaque Assay

    trans -Infection with henipaviruses requires the expression of HS. (A) Lymphocyte-mediated  trans -infection by NiV and HeV. PBLs were incubated with either NiV or HeV, washed, cultured for 24 h, and then transferred to Vero cell monolayers, which were used for the determination of the viral titers after 4 days of coculture, using infectious center assays. (B) CHO-K1 cells, treated or not with heparinase 3, and three HS-deficient CHO lines, pgsA-745, pgsB-619, and pgsD-677, were incubated with NiV and analyzed for their capacities to transmit infection to susceptible Vero cells in  trans . Results are expressed as a percentage of inhibition compared to results with untreated cells ± SD. *,  P

    Journal: mBio

    Article Title: Heparan Sulfate-Dependent Enhancement of Henipavirus Infection

    doi: 10.1128/mBio.02427-14

    Figure Lengend Snippet: trans -Infection with henipaviruses requires the expression of HS. (A) Lymphocyte-mediated trans -infection by NiV and HeV. PBLs were incubated with either NiV or HeV, washed, cultured for 24 h, and then transferred to Vero cell monolayers, which were used for the determination of the viral titers after 4 days of coculture, using infectious center assays. (B) CHO-K1 cells, treated or not with heparinase 3, and three HS-deficient CHO lines, pgsA-745, pgsB-619, and pgsD-677, were incubated with NiV and analyzed for their capacities to transmit infection to susceptible Vero cells in trans . Results are expressed as a percentage of inhibition compared to results with untreated cells ± SD. *, P

    Article Snippet: In some experiments, cells were incubated for 1 h at 37°C with 10 U/ml of heparinase 3 (Sigma) and washed 3 times before contact with NiV.

    Techniques: Infection, Expressing, Incubation, Cell Culture, Inhibition

    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P

    Journal: Cell Adhesion & Migration

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

    doi: 10.1080/19336918.2015.1103421

    Figure Lengend Snippet: Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P

    Article Snippet: Treatment of cells with heparinase I/III and sodium chlorate To assess the influence of surface heparan sulfate digestion on the cell surface expression of Hsp90α and Hsp90β, A-172 and HT1080 cells were washed with DMEM and incubated for 1 h at 37°C with a heparinase I/III blend from Flavobacterium heparinum (Sigma-Aldrich) diluted in DMEM-FBS (0.03 IU/ml).

    Techniques: Incubation, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Negative Control, Expressing

    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

    N-sulfate-enriched microenvironment forms a belt around the demyelinated lesion. HS (10E4) labeling on the contra- ( A ) and ipsi- ( B–C ) lateral side to the lesion illustrates the generation of a N-sulfated microenvironment surrounding the lesion (delimited by white dashed lines) at five dpi (n = 3). No immunoreactivity was found after Heparinase I treatment ( D ) thus validating the 10E4 antibody specificity. Scale bars: 20 µm in A, B, D; 10 µm in C. CC, corpus callosum; V, ventricle.

    Journal: eLife

    Article Title: Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production

    doi: 10.7554/eLife.51735

    Figure Lengend Snippet: N-sulfate-enriched microenvironment forms a belt around the demyelinated lesion. HS (10E4) labeling on the contra- ( A ) and ipsi- ( B–C ) lateral side to the lesion illustrates the generation of a N-sulfated microenvironment surrounding the lesion (delimited by white dashed lines) at five dpi (n = 3). No immunoreactivity was found after Heparinase I treatment ( D ) thus validating the 10E4 antibody specificity. Scale bars: 20 µm in A, B, D; 10 µm in C. CC, corpus callosum; V, ventricle.

    Article Snippet: For N-sulfated motifs labeling, floating sections from PFA perfused-brain were incubated for 2 hr 30 at 37°C in buffer (100 mM Sodium Chloride, 1 mM Calcium Chloride, 50 mM Hepes 5 µg, BSA pH 7) with or without Heparinase (3.3 mU from Flavobacterium heparinum, Seikagaku Kogyo Co. # 100700, Japan) ( ) before permeabilization.

    Techniques: Labeling

    Effects of PD98059 and heparinase on SMC marker gene expression under 3-D interstitial flow. PD98059 and heparinase reverse interstitial flow-induced reductions in SM-MHC, smoothelin, and calponin expression, but further enhance α-SMA and SM22 expression in 3-D. SMCs (A) and MFBs (B) in collagen gels were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to interstitial flow (1 cmH 2 O) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: Effects of PD98059 and heparinase on SMC marker gene expression under 3-D interstitial flow. PD98059 and heparinase reverse interstitial flow-induced reductions in SM-MHC, smoothelin, and calponin expression, but further enhance α-SMA and SM22 expression in 3-D. SMCs (A) and MFBs (B) in collagen gels were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to interstitial flow (1 cmH 2 O) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Marker, Expressing, Flow Cytometry, Quantitative RT-PCR

    Cleavage of heparan sulfate glycosaminglycans (HS-GAGs) by heparinase. SMCs were grown on the plate for 2 days, and then incubated with 6.7 IU/L heparinase III (Hep) for 1 h followed by immunostaining for HS-GAGs. The surfaces of SMCs present abundant HS-GAGs (blue), which was successfully cleaved by heparinase III. Cell nuclei were stained by propidium iodide shown in red.

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: Cleavage of heparan sulfate glycosaminglycans (HS-GAGs) by heparinase. SMCs were grown on the plate for 2 days, and then incubated with 6.7 IU/L heparinase III (Hep) for 1 h followed by immunostaining for HS-GAGs. The surfaces of SMCs present abundant HS-GAGs (blue), which was successfully cleaved by heparinase III. Cell nuclei were stained by propidium iodide shown in red.

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Incubation, Immunostaining, Staining

    PD98059 and heparinase suppress both laminar flow and interstitial flow-induced ERK1/2 activation. Cells in 2-D or 3-D were pretreated with ERK1/2 inhibitor PD98059 (PD) or heparinase III (Hep) and then exposed to laminar flow or interstitial flow for 0 to 30 min. Cells were lysed and proteins were extracted for Western blotting. Gel panels were representative Western blots from three independent experiments, where similar results were found.

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: PD98059 and heparinase suppress both laminar flow and interstitial flow-induced ERK1/2 activation. Cells in 2-D or 3-D were pretreated with ERK1/2 inhibitor PD98059 (PD) or heparinase III (Hep) and then exposed to laminar flow or interstitial flow for 0 to 30 min. Cells were lysed and proteins were extracted for Western blotting. Gel panels were representative Western blots from three independent experiments, where similar results were found.

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Flow Cytometry, Activation Assay, Western Blot

    Effects of PD98059 and heparinase on SMC marker gene expression under 2-D laminar flow. PD98059 and heparinase reverse laminar flow-induced reductions in expression of SMC marker genes in 2-D. SMCs (A) and MFBs (B) were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to 8 dyn/cm 2 laminar shear stress for 15 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: Effects of PD98059 and heparinase on SMC marker gene expression under 2-D laminar flow. PD98059 and heparinase reverse laminar flow-induced reductions in expression of SMC marker genes in 2-D. SMCs (A) and MFBs (B) were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to 8 dyn/cm 2 laminar shear stress for 15 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Marker, Expressing, Flow Cytometry, Quantitative RT-PCR

    GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K d s were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125 I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with heparinase III (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125 I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.

    Journal: The Journal of Cell Biology

    Article Title: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

    doi: 10.1083/jcb.201009136

    Figure Lengend Snippet: GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K d s were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125 I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with heparinase III (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125 I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.

    Article Snippet: Heparinase III was obtained from Seikagaku Corp. (Seikagaku’s trademark for this product is Heparitinase I).

    Techniques: Binding Assay, SPR Assay, Concentration Assay, Immunoprecipitation, Planar Chromatography, Negative Control, Silver Staining, Western Blot, SDS Page, Transfection, Construct

    GDNF, NRTN, and ARTN induce SHEP cell adherence and SFK activation in an HSPG-dependent manner. (A) RT-PCR of the cDNA from SHEP cells with syndecan-3–specific primers. The resulting 140-bp band represents a syndecan-3 mRNA-derived PCR product (lane 2). (lane 1) Molecular weight markers with sizes in bp. (B) Crystal violet staining of SHEP cells plated on immobilized GDNF, ARTN, NRTN, and PSPN with or without heparinase III (H’aseIII) pretreatment. (C) Quantification of attached SHEP cells on immobilized GFLs. Some cells were treated with heparinase III. Quantification also includes controls with cell attachment on BSA and attachment of cells treated with heparinase III on tissue culture plates. (D) Quantification of SHEP cell adherence to immobilized GDNF and ΔN-GDNF. Cells were plated on GDNF untreated, or they were pretreated with PI-PLC, chondroitinase ABC (Ch’ase ABC), NCAM function-blocking antibodies (NCAM BLK), or GDNF function-blocking antibodies (GDNF BLK). (C and D) Error bars show SEM from three independent assays. (E) Western blot (WB) for activated SFK in lysates from SHEP cells plated on GDNF, ΔN-GDNF, or BSA. In control experiments, cells plated on GDNF were treated with heparinase III (HIII) or PI-PLC. Western blots were probed with anti-pY 418 Src (top) or anti-Src (bottom) antibodies. (F) SHEP cell adherence and spreading on immobilized GDNF in the absence or presence of 2 µM SFK inhibitor SU6656. Cell spreading on immobilized GDNF was impaired in the presence of the inhibitor, whereas adherence of SHEP cells was not significantly affected by SU6656. (G) Adherence and spreading of SHEP cells infected with adenovirus expressing GFP (AdGFP) or dominant-negative Src (AdDN-Src/GFP) on immobilized GDNF. Transduction of cells with AdDN-Src/GFP resulted in SHEP’s failure to spread on immobilized GDNF. GFP-expressing adenovirus did not affect SHEP cell spreading on the GDNF matrix. Untreated, UNTR. Bars, 100 µM.

    Journal: The Journal of Cell Biology

    Article Title: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

    doi: 10.1083/jcb.201009136

    Figure Lengend Snippet: GDNF, NRTN, and ARTN induce SHEP cell adherence and SFK activation in an HSPG-dependent manner. (A) RT-PCR of the cDNA from SHEP cells with syndecan-3–specific primers. The resulting 140-bp band represents a syndecan-3 mRNA-derived PCR product (lane 2). (lane 1) Molecular weight markers with sizes in bp. (B) Crystal violet staining of SHEP cells plated on immobilized GDNF, ARTN, NRTN, and PSPN with or without heparinase III (H’aseIII) pretreatment. (C) Quantification of attached SHEP cells on immobilized GFLs. Some cells were treated with heparinase III. Quantification also includes controls with cell attachment on BSA and attachment of cells treated with heparinase III on tissue culture plates. (D) Quantification of SHEP cell adherence to immobilized GDNF and ΔN-GDNF. Cells were plated on GDNF untreated, or they were pretreated with PI-PLC, chondroitinase ABC (Ch’ase ABC), NCAM function-blocking antibodies (NCAM BLK), or GDNF function-blocking antibodies (GDNF BLK). (C and D) Error bars show SEM from three independent assays. (E) Western blot (WB) for activated SFK in lysates from SHEP cells plated on GDNF, ΔN-GDNF, or BSA. In control experiments, cells plated on GDNF were treated with heparinase III (HIII) or PI-PLC. Western blots were probed with anti-pY 418 Src (top) or anti-Src (bottom) antibodies. (F) SHEP cell adherence and spreading on immobilized GDNF in the absence or presence of 2 µM SFK inhibitor SU6656. Cell spreading on immobilized GDNF was impaired in the presence of the inhibitor, whereas adherence of SHEP cells was not significantly affected by SU6656. (G) Adherence and spreading of SHEP cells infected with adenovirus expressing GFP (AdGFP) or dominant-negative Src (AdDN-Src/GFP) on immobilized GDNF. Transduction of cells with AdDN-Src/GFP resulted in SHEP’s failure to spread on immobilized GDNF. GFP-expressing adenovirus did not affect SHEP cell spreading on the GDNF matrix. Untreated, UNTR. Bars, 100 µM.

    Article Snippet: Heparinase III was obtained from Seikagaku Corp. (Seikagaku’s trademark for this product is Heparitinase I).

    Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Molecular Weight, Staining, Cell Attachment Assay, Planar Chromatography, Blocking Assay, Western Blot, Infection, Expressing, Dominant Negative Mutation, Transduction

    Immobilized GDNF induces neurite outgrowth in rat embryonic hippocampal neurons. (A) Neurite outgrowth in E17 rat hippocampal neurons on immobilized GDNF. Neurons are stained with tubulin-βIII antibodies. Bar, 20 µM. (B) Neurite outgrowth in hippocampal neurons plated on BSA. Neurons are stained with tubulin-βIII antibodies. (C) Quantification of neurite outgrowth on immobilized GDNF, ΔN-GDNF, PSPN, and BSA. Neurons plated on GDNF were untreated (UNTR) or preincubated with heparinase III (H’aseIII), PI-PLC, or 2 µM SFK inhibitor SU6656. As a control, soluble GDNF was added to neurons grown on BSA. Error bars show SEM from three to five independent experiments (*, P

    Journal: The Journal of Cell Biology

    Article Title: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

    doi: 10.1083/jcb.201009136

    Figure Lengend Snippet: Immobilized GDNF induces neurite outgrowth in rat embryonic hippocampal neurons. (A) Neurite outgrowth in E17 rat hippocampal neurons on immobilized GDNF. Neurons are stained with tubulin-βIII antibodies. Bar, 20 µM. (B) Neurite outgrowth in hippocampal neurons plated on BSA. Neurons are stained with tubulin-βIII antibodies. (C) Quantification of neurite outgrowth on immobilized GDNF, ΔN-GDNF, PSPN, and BSA. Neurons plated on GDNF were untreated (UNTR) or preincubated with heparinase III (H’aseIII), PI-PLC, or 2 µM SFK inhibitor SU6656. As a control, soluble GDNF was added to neurons grown on BSA. Error bars show SEM from three to five independent experiments (*, P

    Article Snippet: Heparinase III was obtained from Seikagaku Corp. (Seikagaku’s trademark for this product is Heparitinase I).

    Techniques: Staining, Planar Chromatography

    Both HS and protein components of SCM are required for boundary formation Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); heparinase treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75 NTR (green). The number of cells mingling with astrocytes was counted across a 300 μm line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate ± SEM. Scale bar 50 μm. ** p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation

    doi: 10.1523/JNEUROSCI.6340-11.2012

    Figure Lengend Snippet: Both HS and protein components of SCM are required for boundary formation Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); heparinase treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75 NTR (green). The number of cells mingling with astrocytes was counted across a 300 μm line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate ± SEM. Scale bar 50 μm. ** p

    Article Snippet: HS was digested by the addition of 10 mU each of heparinase I (EC 4.2.2.7), II (EC number not assigned) and III (EC 4.2.2.8) (Ibex Technologies, Montreal, Canada) to 4 ml SCM, followed by incubation at 37°C for 6 h. A further 10 mU of each heparinase enzyme was added to SCM and the reaction incubated overnight at 37°C.

    Techniques: Staining, Activity Assay

    SAX-HPLC analysis of fluorescently labelled HS disaccharides purified from OCM and SCM indicate that SCs and OECs secrete distinct HS structures HS purified from OCM (A) and SCM (B) was digested with heparinase enzymes to generate HS disaccharides, which were fluorescently labelled with BODIPY and separated by SAX-HPLC over a 45 minute, 0-1.5 M NaCl gradient, (A and B, black lines). HS disaccharide standards were separated over the same gradient as a reference guide (A and B, dashed lines). Relative abundances of the eight HS standard disaccharides in each conditioned medium sample were calculated as a percentage of total HS (C). A summary table detailing the composition of OCM HS and SCM HS is also presented (D). SCM HS was found to be more highly sulfated than OCM HS, with an average of 1.02 sulfates per disaccharide compared to 0.75 sulfates per disaccharide in OCM HS (D). SCM HS contained a higher proportion of di- and tri-sulfated disaccharides (disaccharides 4, 5, 8 and 6) and the singly 6-O-sulfated disaccharide (disaccharide 2) compared to OCM HS (C), which contained a higher proportion of the unsulfated disaccharide (disaccharide 1) (C, D). (UA) uronic acid, (GlcN) glucosamine, (NAc) N-acetyl, (NS) N-sulfate, (6OS) 6-O-sulfate, (2OS) 2-O-sulfate.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation

    doi: 10.1523/JNEUROSCI.6340-11.2012

    Figure Lengend Snippet: SAX-HPLC analysis of fluorescently labelled HS disaccharides purified from OCM and SCM indicate that SCs and OECs secrete distinct HS structures HS purified from OCM (A) and SCM (B) was digested with heparinase enzymes to generate HS disaccharides, which were fluorescently labelled with BODIPY and separated by SAX-HPLC over a 45 minute, 0-1.5 M NaCl gradient, (A and B, black lines). HS disaccharide standards were separated over the same gradient as a reference guide (A and B, dashed lines). Relative abundances of the eight HS standard disaccharides in each conditioned medium sample were calculated as a percentage of total HS (C). A summary table detailing the composition of OCM HS and SCM HS is also presented (D). SCM HS was found to be more highly sulfated than OCM HS, with an average of 1.02 sulfates per disaccharide compared to 0.75 sulfates per disaccharide in OCM HS (D). SCM HS contained a higher proportion of di- and tri-sulfated disaccharides (disaccharides 4, 5, 8 and 6) and the singly 6-O-sulfated disaccharide (disaccharide 2) compared to OCM HS (C), which contained a higher proportion of the unsulfated disaccharide (disaccharide 1) (C, D). (UA) uronic acid, (GlcN) glucosamine, (NAc) N-acetyl, (NS) N-sulfate, (6OS) 6-O-sulfate, (2OS) 2-O-sulfate.

    Article Snippet: HS was digested by the addition of 10 mU each of heparinase I (EC 4.2.2.7), II (EC number not assigned) and III (EC 4.2.2.8) (Ibex Technologies, Montreal, Canada) to 4 ml SCM, followed by incubation at 37°C for 6 h. A further 10 mU of each heparinase enzyme was added to SCM and the reaction incubated overnight at 37°C.

    Techniques: High Performance Liquid Chromatography, Purification

    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:

    Posterior mouse extracellular matrix (ECM) induces pattern formation by axolotl blastema cells. (A) Schematic diagram of surgical procedures. Mouse hindlimb (femur) skin samples (red) were collected from mice for ECM extraction followed by grafting into an axolotl skin wound with a deviated brachial nerve (blue). (B) Differential regenerative responses to grafts of mouse‐derived anterior or posterior ECM from skin samples at postnatal day 3−4 either with or without heparin lyase III treatment. Ant, anterior; Post, posterior; (−)HS, heparin lyase III treatment to remove heparan sulfate from the ECM. (C) Whole‐mount skeletal preparation of a skeletal‐like element formed by axolotl blastema cells in response to an ECM graft from the posterior of a mouse limb at about 2 months post‐grafting: red, ossified bone; blue, cartilage indicated by arrow.

    Journal: Regeneration

    Article Title: Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum)

    doi: 10.1002/reg2.40

    Figure Lengend Snippet: Posterior mouse extracellular matrix (ECM) induces pattern formation by axolotl blastema cells. (A) Schematic diagram of surgical procedures. Mouse hindlimb (femur) skin samples (red) were collected from mice for ECM extraction followed by grafting into an axolotl skin wound with a deviated brachial nerve (blue). (B) Differential regenerative responses to grafts of mouse‐derived anterior or posterior ECM from skin samples at postnatal day 3−4 either with or without heparin lyase III treatment. Ant, anterior; Post, posterior; (−)HS, heparin lyase III treatment to remove heparan sulfate from the ECM. (C) Whole‐mount skeletal preparation of a skeletal‐like element formed by axolotl blastema cells in response to an ECM graft from the posterior of a mouse limb at about 2 months post‐grafting: red, ossified bone; blue, cartilage indicated by arrow.

    Article Snippet: Heparan sulfate moieties were removed enzymatically by incubating decellularized ECM preparations in 0.008 IU/mL heparin lyase III (Seikagaku) for 4 h at 37°C, after which they were rinsed in PBS and stored in PBS at 4°C until grafted.

    Techniques: Mouse Assay, Derivative Assay

    Heparan sulfate (HS) mediate position‐specific regulation of blastema formation and pattern formation during regeneration. (A) Differential regenerative responses to grafts of anterior/posterior extracellular matrix (ECM) with or without heparin lyase III (HepIII) treatment: Ant, anterior; Post, posterior; (−)HS, HepIII treated to remove HS from the ECM. (B) Dose‐dependent induction of pattern formation by grafts of artificial ECM containing low HS (2.5 mg/mL), med HS (5 mg/mL) or high HS (10 mg/mL) (Sigma, porcine intestinal mucosa).

    Journal: Regeneration

    Article Title: Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum)

    doi: 10.1002/reg2.40

    Figure Lengend Snippet: Heparan sulfate (HS) mediate position‐specific regulation of blastema formation and pattern formation during regeneration. (A) Differential regenerative responses to grafts of anterior/posterior extracellular matrix (ECM) with or without heparin lyase III (HepIII) treatment: Ant, anterior; Post, posterior; (−)HS, HepIII treated to remove HS from the ECM. (B) Dose‐dependent induction of pattern formation by grafts of artificial ECM containing low HS (2.5 mg/mL), med HS (5 mg/mL) or high HS (10 mg/mL) (Sigma, porcine intestinal mucosa).

    Article Snippet: Heparan sulfate moieties were removed enzymatically by incubating decellularized ECM preparations in 0.008 IU/mL heparin lyase III (Seikagaku) for 4 h at 37°C, after which they were rinsed in PBS and stored in PBS at 4°C until grafted.

    Techniques: