heparinase iii  (Millipore)


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  • 99
    Name:
    Heparinase III from Flavobacterium heparinum
    Description:

    Catalog Number:
    h8891
    Price:
    None
    Applications:
    Heparinase III from Flavobacterium heparinum has been used to cleave specific GAGs. It has been used to study its effect on the electrophoretic mobility of the protein reacting with the anti-aFGF (acidic fibroblast growth factor) antibody.
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    Structured Review

    Millipore heparinase iii
    Heparinase III from Flavobacterium heparinum

    https://www.bioz.com/result/heparinase iii/product/Millipore
    Average 99 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    heparinase iii - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "A Biomechanical Role for Perlecan in the Pericellular Matrix of Articular Cartilage"

    Article Title: A Biomechanical Role for Perlecan in the Pericellular Matrix of Articular Cartilage

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2012.05.002

    Effect of heparinase III digestion on ECM composition and micromechanical properties. (A) Histological staining with safranin-O (red, proteoglycans) and fast green (blue, collagens) demonstrated that digestion with heparinase III had a minimal effect
    Figure Legend Snippet: Effect of heparinase III digestion on ECM composition and micromechanical properties. (A) Histological staining with safranin-O (red, proteoglycans) and fast green (blue, collagens) demonstrated that digestion with heparinase III had a minimal effect

    Techniques Used: Staining

    Immunofluorescence labeling of unfixed porcine cartilage sections following heparinase III. Dual IF labeling of (A) HS epitope 3G10 and (B) perlecan exhibited a nearly one-to-one overlay (C). (D) 3G10 and (E) type VI collagen co-localized in the pericellular
    Figure Legend Snippet: Immunofluorescence labeling of unfixed porcine cartilage sections following heparinase III. Dual IF labeling of (A) HS epitope 3G10 and (B) perlecan exhibited a nearly one-to-one overlay (C). (D) 3G10 and (E) type VI collagen co-localized in the pericellular

    Techniques Used: Immunofluorescence, Labeling

    Effect of heparinase III digestion on PCM micromechanical properties. (A) PCM moduli as defined by perlecan significantly increased with heparinase III digestion (a: p
    Figure Legend Snippet: Effect of heparinase III digestion on PCM micromechanical properties. (A) PCM moduli as defined by perlecan significantly increased with heparinase III digestion (a: p

    Techniques Used:

    2) Product Images from "Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction"

    Article Title: Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction

    Journal: The FASEB Journal

    doi: 10.1096/fj.10-168971

    Disruption of HSPGs in mESCs. A ) Two schemes were used for disrupting HSPG function: NaClO 3 blocks sulfation of the heparan sulfate chains, while heparinase III cleaves heparan sulfate chains from the core glycoproteins. B , C ) Gene expression of mESCs
    Figure Legend Snippet: Disruption of HSPGs in mESCs. A ) Two schemes were used for disrupting HSPG function: NaClO 3 blocks sulfation of the heparan sulfate chains, while heparinase III cleaves heparan sulfate chains from the core glycoproteins. B , C ) Gene expression of mESCs

    Techniques Used: Expressing

    3) Product Images from "Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans"

    Article Title: Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans

    Journal: Scientific Reports

    doi: 10.1038/srep34537

    Analysis of perlecan GAG-composition with SDS-PAGE. ( a ) Schematic illustration of perlecan. The proteoglycan may be substituted with GAGs at various positions, including three HS-chains at the N-terminal end as well as a CS-chain at the C-terminal end 18 . The number of disaccharides (n) and the degree of sulfation may vary at each GAG-position. ( b ) Trypsin-digested mouse perlecan was applied onto a SAX-column and enriched for GAG-glycopeptides (see Methods). The GAG-glycopeptides were eluted from the column with increasing salt concentrations (0.4 M, 0. 8 M and 1.6 M NaCl) and analyzed with SDS-PAGE / Alcian blue. Perlecan incubated with and without trypsin was loaded as control. ( c , d ) An identical set of SAX-enriched GAG-glycopeptides were treated with either chondroitinase ABC (CSase) ( c ) or heparinase (HSase) ( d ) prior to the SDS-PAGE / Alcian blue analysis.
    Figure Legend Snippet: Analysis of perlecan GAG-composition with SDS-PAGE. ( a ) Schematic illustration of perlecan. The proteoglycan may be substituted with GAGs at various positions, including three HS-chains at the N-terminal end as well as a CS-chain at the C-terminal end 18 . The number of disaccharides (n) and the degree of sulfation may vary at each GAG-position. ( b ) Trypsin-digested mouse perlecan was applied onto a SAX-column and enriched for GAG-glycopeptides (see Methods). The GAG-glycopeptides were eluted from the column with increasing salt concentrations (0.4 M, 0. 8 M and 1.6 M NaCl) and analyzed with SDS-PAGE / Alcian blue. Perlecan incubated with and without trypsin was loaded as control. ( c , d ) An identical set of SAX-enriched GAG-glycopeptides were treated with either chondroitinase ABC (CSase) ( c ) or heparinase (HSase) ( d ) prior to the SDS-PAGE / Alcian blue analysis.

    Techniques Used: SDS Page, Incubation

    4) Product Images from "Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells"

    Article Title: Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.23302

    Immunofluorescence staining for HA in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HA. Treatment with heparinase III did not change the intensity of HA fluorescence
    Figure Legend Snippet: Immunofluorescence staining for HA in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HA. Treatment with heparinase III did not change the intensity of HA fluorescence

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Fluorescence

    Immunofluorescence staining for HSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HSPG. Treatment with heparinase III significantly reduced expression of HSPG
    Figure Legend Snippet: Immunofluorescence staining for HSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HSPG. Treatment with heparinase III significantly reduced expression of HSPG

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Expressing

    ESC-derived EC gene response to shear stress (5 dyn/cm 2 for 8h) after treatment with heparinase III (20 mU/ml). Relative gene expression (gene/GAPDH) was measured by qPCR and normalized to its own static control case prior to enzyme treatment. Heparinase
    Figure Legend Snippet: ESC-derived EC gene response to shear stress (5 dyn/cm 2 for 8h) after treatment with heparinase III (20 mU/ml). Relative gene expression (gene/GAPDH) was measured by qPCR and normalized to its own static control case prior to enzyme treatment. Heparinase

    Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

    Immunofluorescence staining for CSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain CSPG. Treatment with heparinase III did not change the intensity of CSPG fluorescence
    Figure Legend Snippet: Immunofluorescence staining for CSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain CSPG. Treatment with heparinase III did not change the intensity of CSPG fluorescence

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Fluorescence

    5) Product Images from "Phospholipase A2 Mediates Apolipoprotein-Independent Uptake of Chylomicron Remnant-Like Particles by Human Macrophages"

    Article Title: Phospholipase A2 Mediates Apolipoprotein-Independent Uptake of Chylomicron Remnant-Like Particles by Human Macrophages

    Journal: International Journal of Vascular Medicine

    doi: 10.1155/2012/501954

    Role of proteoglycan-mediated bridging in macrophage lipid accumulation. HMDM were incubated 1 h in serum-free medium before treatment with 33 U/mL heparinase III (Hep III) or 33 U/mL heparinase I (Hep I) in combination with 50 mM Na chlorate. Both untreated and treated macrophages were then incubated 24 h with 80 μ g cholesterol/mL CRLPw/o. Treatments were continued during the incubations. Macrophage triacylglycerol (TG) and total cholesterol (TCH) contents are reported as nmol/mg protein (means ± SD, n = 4). * P
    Figure Legend Snippet: Role of proteoglycan-mediated bridging in macrophage lipid accumulation. HMDM were incubated 1 h in serum-free medium before treatment with 33 U/mL heparinase III (Hep III) or 33 U/mL heparinase I (Hep I) in combination with 50 mM Na chlorate. Both untreated and treated macrophages were then incubated 24 h with 80 μ g cholesterol/mL CRLPw/o. Treatments were continued during the incubations. Macrophage triacylglycerol (TG) and total cholesterol (TCH) contents are reported as nmol/mg protein (means ± SD, n = 4). * P

    Techniques Used: Incubation

    6) Product Images from "Virion-associated viral fibroblast growth factor stimulates cell motility"

    Article Title: Virion-associated viral fibroblast growth factor stimulates cell motility

    Journal: Virology

    doi: 10.1016/j.virol.2009.09.011

    Virus induced cell migration. (A) SF-21 cells (2 × 10 4 ) were placed in the upper chamber of a migration chamber with an 8 μM pore size and purified infectious virions from AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA, were placed in the lower chamber. After 4 hours of incubation at 27 °C, the upper chamber was removed and the number of cells that migrated to the lower chamber was determined using Cell Titer Glo Luminescent Assay. (B) AcBAC-HSP70vFGFHA virions were treated with heparinase III to release vFGF-heparan complexes from the virus envelope. Heparinase III-released vFGF into the supernatant or heparinase III-treated and -untreated virions were used in motility assays. (***p
    Figure Legend Snippet: Virus induced cell migration. (A) SF-21 cells (2 × 10 4 ) were placed in the upper chamber of a migration chamber with an 8 μM pore size and purified infectious virions from AcBAC-vfgfHARep, AcBAC-vfgfKO, or AcBAC-HSP70vfgfHA, were placed in the lower chamber. After 4 hours of incubation at 27 °C, the upper chamber was removed and the number of cells that migrated to the lower chamber was determined using Cell Titer Glo Luminescent Assay. (B) AcBAC-HSP70vFGFHA virions were treated with heparinase III to release vFGF-heparan complexes from the virus envelope. Heparinase III-released vFGF into the supernatant or heparinase III-treated and -untreated virions were used in motility assays. (***p

    Techniques Used: Migration, Purification, Incubation, Luminescence Assay

    7) Product Images from "Heparan Sulfate Proteoglycans Are Ligands for Receptor Protein Tyrosine Phosphatase ?"

    Article Title: Heparan Sulfate Proteoglycans Are Ligands for Receptor Protein Tyrosine Phosphatase ?

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.6.1881-1892.2002

    Müller glia endfeet express two classes of cPTPσ ligands. Retinal basal laminae with glial endfeet attached were flat mounted and probed by RAP in situ with conditioned media containing cPTPσ1-AP (A to E), FN3Δ-AP (F), M1 (G), M2 (H), M3 (I), M4 (J), M5 (K), and M6 (L). Heparin addition (B) or heparinase III pretreatment (C) reduces binding compared to control treatments (A and F) but does not abolish it. cPTPσ mutants with impaired heparin-binding properties also show reduced binding on glial endfeet (H to J). Chondroitin sulfate addition (D) or chondroitinase ABC pretreatment (E) does not affect cPTPσ binding significantly. Arrows indicate ringlike endfeet. Scale bar, 10 μm.
    Figure Legend Snippet: Müller glia endfeet express two classes of cPTPσ ligands. Retinal basal laminae with glial endfeet attached were flat mounted and probed by RAP in situ with conditioned media containing cPTPσ1-AP (A to E), FN3Δ-AP (F), M1 (G), M2 (H), M3 (I), M4 (J), M5 (K), and M6 (L). Heparin addition (B) or heparinase III pretreatment (C) reduces binding compared to control treatments (A and F) but does not abolish it. cPTPσ mutants with impaired heparin-binding properties also show reduced binding on glial endfeet (H to J). Chondroitin sulfate addition (D) or chondroitinase ABC pretreatment (E) does not affect cPTPσ binding significantly. Arrows indicate ringlike endfeet. Scale bar, 10 μm.

    Techniques Used: In Situ, Binding Assay

    Binding of cPTPσ to the E6 chick retinal BL is mediated by HS chains. Results of receptor affinity probe assays using the extracellular region of cPTPσ1 fused to alkaline phosphatase are shown. Retina sections were untreated (A to D) or pretreated with heparinase III (E) or chondroitinase ABC (F). cPTPσ1-AP conditioned medium was used alone (A) or preincubated with heparin (B), HS (C), or chondroitin sulfate (D). The BL staining indicates cPTPσ1-AP binding. Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.
    Figure Legend Snippet: Binding of cPTPσ to the E6 chick retinal BL is mediated by HS chains. Results of receptor affinity probe assays using the extracellular region of cPTPσ1 fused to alkaline phosphatase are shown. Retina sections were untreated (A to D) or pretreated with heparinase III (E) or chondroitinase ABC (F). cPTPσ1-AP conditioned medium was used alone (A) or preincubated with heparin (B), HS (C), or chondroitin sulfate (D). The BL staining indicates cPTPσ1-AP binding. Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

    Techniques Used: Binding Assay, Staining

    cPTPσ binds agrin and collagen XVIII via their HS chains. An HSPG-enriched fraction from chicken embryo vitreous bodies (A), purified agrin (B), and purified collagen XVIII (C) samples, separated by SDS-6% polyacrylamide gel electrophoresis, were transferred to nitrocellulose and probed with antibodies specific for agrin or collagen XVIII or were incubated with cPTPσ1-VSV conditioned medium. cPTPσ1-VSV was detected using antibody to VSV. Samples were either not treated with enzymes (lanes a, e, and i) or predigested with heparinase III (lanes b, f, and j), collagenase (lanes c, g, and k), or chondroitinase ABC (lanes d, h, and l). Arrows in panel A indicate the two bands corresponding in molecular mass to agrin (upper arrow) and collagen XVIII (lower arrow) observed in the blot overlay assay.
    Figure Legend Snippet: cPTPσ binds agrin and collagen XVIII via their HS chains. An HSPG-enriched fraction from chicken embryo vitreous bodies (A), purified agrin (B), and purified collagen XVIII (C) samples, separated by SDS-6% polyacrylamide gel electrophoresis, were transferred to nitrocellulose and probed with antibodies specific for agrin or collagen XVIII or were incubated with cPTPσ1-VSV conditioned medium. cPTPσ1-VSV was detected using antibody to VSV. Samples were either not treated with enzymes (lanes a, e, and i) or predigested with heparinase III (lanes b, f, and j), collagenase (lanes c, g, and k), or chondroitinase ABC (lanes d, h, and l). Arrows in panel A indicate the two bands corresponding in molecular mass to agrin (upper arrow) and collagen XVIII (lower arrow) observed in the blot overlay assay.

    Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Incubation, Overlay Assay

    8) Product Images from "Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum"

    Article Title: Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum

    Journal: The Journal of Experimental Medicine

    doi:

    Effect of GAGs on P. falciparum rosetting. A shows disruption of rosettes exerted by different GAGs. FCR3S1.2 cultures were incubated with GAGs for 1 h at 37°C and compared to control culture. Results are the means and standard error of three separate experiments. B shows the effect of enzyme treatment of uninfected, C-FDA–labeled erythrocytes in a competitive assay of rosette reformation in the presence of normal erythrocytes and FCR3S1.2-infected pRBCs. Results are the means and standard error of three separate experiments, or two experiments for neuraminidase. Neuraminidase and chondroitinase ABC concentrations are in IU, whereas the heparinase III concentration is in Sigma units. (One Sigma unit corresponds to ∼1.7 × 10 −3 IU.)
    Figure Legend Snippet: Effect of GAGs on P. falciparum rosetting. A shows disruption of rosettes exerted by different GAGs. FCR3S1.2 cultures were incubated with GAGs for 1 h at 37°C and compared to control culture. Results are the means and standard error of three separate experiments. B shows the effect of enzyme treatment of uninfected, C-FDA–labeled erythrocytes in a competitive assay of rosette reformation in the presence of normal erythrocytes and FCR3S1.2-infected pRBCs. Results are the means and standard error of three separate experiments, or two experiments for neuraminidase. Neuraminidase and chondroitinase ABC concentrations are in IU, whereas the heparinase III concentration is in Sigma units. (One Sigma unit corresponds to ∼1.7 × 10 −3 IU.)

    Techniques Used: Incubation, Labeling, Infection, Concentration Assay

    9) Product Images from "RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts"

    Article Title: RANTES/CCL5 Induces Collagen Degradation by Activating MMP-1 and MMP-13 Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01341

    Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) contributes to the proinflammatory cytokine-mediated matrix metalloproteinase (MMP)-1 and MMP-13 expression in rheumatoid arthritis (RA). (A) Met-RANTES inhibits interleukin (IL)-1β-induced MMP-1 and MMP-13 production in rheumatoid arthritis synovial fibroblasts (RASFs). (B) Effect of RANTES/CCL5 knockdown on IL-1β-induced MMP-1 and MMP-13 production in RASFs. RASFs were transfected with RANTES/CCL5-specific small-interfering RNA (siRNA) for 48 h and then stimulated with IL-1β (5 ng/ml) for 24 h. Levels of MMP-1 and MMP-13 in conditioned media was quantified using ELISA and represented as percent of IL-1β-treatment control. (C) Conditioned media from the above treated samples were also evaluated to determine percent knockdown of RANTES/CCL5 using siRNA approach and represented as percent of IL-1β treatment control. (D) Degradation of heparan sulfate proteoglycans (HSPG) by heparinase III reduces RANTES/CCL5-induced MMP-1 and MMP-13 expression in RASFs. RASFs were pretreated with heparinase III (0.5 U/ml) for 2 h followed by stimulation with RANTES/CCL5 (100 ng/ml) for 24 h. Conditioned media was concentrated and used to detect MMP-1 and MMP-13 expression using Western immunoblotting. Densitometry was performed to determine the relative changes. (E) RASFs pretreated with heparinase III (0.5 U/ml) for 2 h followed by stimulation with RANTES/CCL5 (100 ng/ml) for 1 h were used with or without cross-linking step for the detection of RANTES/CCL5 in the whole cell lysates (25 µl). Values are represented as mean ± SE from three independent experiments performed using RASFs from different donors under similar conditions. ## p
    Figure Legend Snippet: Regulated on activation, normal T expressed, and secreted (RANTES)/CC ligand 5 (CCL5) contributes to the proinflammatory cytokine-mediated matrix metalloproteinase (MMP)-1 and MMP-13 expression in rheumatoid arthritis (RA). (A) Met-RANTES inhibits interleukin (IL)-1β-induced MMP-1 and MMP-13 production in rheumatoid arthritis synovial fibroblasts (RASFs). (B) Effect of RANTES/CCL5 knockdown on IL-1β-induced MMP-1 and MMP-13 production in RASFs. RASFs were transfected with RANTES/CCL5-specific small-interfering RNA (siRNA) for 48 h and then stimulated with IL-1β (5 ng/ml) for 24 h. Levels of MMP-1 and MMP-13 in conditioned media was quantified using ELISA and represented as percent of IL-1β-treatment control. (C) Conditioned media from the above treated samples were also evaluated to determine percent knockdown of RANTES/CCL5 using siRNA approach and represented as percent of IL-1β treatment control. (D) Degradation of heparan sulfate proteoglycans (HSPG) by heparinase III reduces RANTES/CCL5-induced MMP-1 and MMP-13 expression in RASFs. RASFs were pretreated with heparinase III (0.5 U/ml) for 2 h followed by stimulation with RANTES/CCL5 (100 ng/ml) for 24 h. Conditioned media was concentrated and used to detect MMP-1 and MMP-13 expression using Western immunoblotting. Densitometry was performed to determine the relative changes. (E) RASFs pretreated with heparinase III (0.5 U/ml) for 2 h followed by stimulation with RANTES/CCL5 (100 ng/ml) for 1 h were used with or without cross-linking step for the detection of RANTES/CCL5 in the whole cell lysates (25 µl). Values are represented as mean ± SE from three independent experiments performed using RASFs from different donors under similar conditions. ## p

    Techniques Used: Activation Assay, Expressing, Transfection, Small Interfering RNA, Enzyme-linked Immunosorbent Assay, Western Blot

    10) Product Images from "Combinatorial Targeting of the Macropinocytotic Pathway in Leukemia and Lymphoma Cells *"

    Article Title: Combinatorial Targeting of the Macropinocytotic Pathway in Leukemia and Lymphoma Cells *

    Journal:

    doi: 10.1074/jbc.M708849200

    Differential effects of heparin, heparinase III, and trypsin on cellular uptake of CAYHRLRRC and tat peptides. Molt-4 ( A and B ) or K562 ( C and D ) cells were incubated with or without heparin (1 mg/ml), heparinase III (0.5 units/ml), or trypsin (1 mg/ml)
    Figure Legend Snippet: Differential effects of heparin, heparinase III, and trypsin on cellular uptake of CAYHRLRRC and tat peptides. Molt-4 ( A and B ) or K562 ( C and D ) cells were incubated with or without heparin (1 mg/ml), heparinase III (0.5 units/ml), or trypsin (1 mg/ml)

    Techniques Used: Incubation

    11) Product Images from "Dendritic space-filling requires a neuronal type-specific extracellular permissive signal in Drosophila"

    Article Title: Dendritic space-filling requires a neuronal type-specific extracellular permissive signal in Drosophila

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

    doi: 10.1073/pnas.1707467114

    Effects of knockdown of HS synthesis genes on dendrite morphology, HS level, and epithelial morphology. ( A – C ) DdaC dendritic fields of a Gal4 A58 control animal ( A ) and animals expressing Gal4 A58 > ttv RNAi ( B ) and Gal4 A58 > sfl RNAi ( C ). ( D – F ) Quantification of total dendrite density ( D ), terminal dendrite density ( E ), and terminal dendrite number ( F ) of control and RNAi animals. *** P ≤ 0.001; ns, not significant; one-way analysis of variance and Tukey’s HSD test. For all quantifications, each circle represents an individual neuron. The number of neurons for each genotype is indicated. The black bars represent the mean, and the red bars represent the SD. ( G – H ′′) Immunostaining of HS in a Gal4 hh > GFP control animal ( G – G ′′) and an animal expressing Gal4 hh > ttv RNAi ( H – H ′′). HS was stained with the antibody 3G10, which recognizes heparinase III-digested HS. ( I – L ′) Expression of E-Cad ( I and I ′), Nrg-GFP ( J and J ′), Mys ( K and K ′), and Vkg-GFP ( L and L ′) in animals expressing Gal4 hh > ttv RNAi ( I , I ′, K , K ′, L , and L ′) and Gal4 hh > sotv RNAi ( J and J ′). E-Cad and Mys expressions were detected by antibody staining, and Nrg-GFP and Vkg-GFP expressions were detected using GFP trap lines. A cross-section for each marker is shown below each 2D projection image. Blue dotted lines indicate locations of cross-sections. (Scale bars, 50 µm.)
    Figure Legend Snippet: Effects of knockdown of HS synthesis genes on dendrite morphology, HS level, and epithelial morphology. ( A – C ) DdaC dendritic fields of a Gal4 A58 control animal ( A ) and animals expressing Gal4 A58 > ttv RNAi ( B ) and Gal4 A58 > sfl RNAi ( C ). ( D – F ) Quantification of total dendrite density ( D ), terminal dendrite density ( E ), and terminal dendrite number ( F ) of control and RNAi animals. *** P ≤ 0.001; ns, not significant; one-way analysis of variance and Tukey’s HSD test. For all quantifications, each circle represents an individual neuron. The number of neurons for each genotype is indicated. The black bars represent the mean, and the red bars represent the SD. ( G – H ′′) Immunostaining of HS in a Gal4 hh > GFP control animal ( G – G ′′) and an animal expressing Gal4 hh > ttv RNAi ( H – H ′′). HS was stained with the antibody 3G10, which recognizes heparinase III-digested HS. ( I – L ′) Expression of E-Cad ( I and I ′), Nrg-GFP ( J and J ′), Mys ( K and K ′), and Vkg-GFP ( L and L ′) in animals expressing Gal4 hh > ttv RNAi ( I , I ′, K , K ′, L , and L ′) and Gal4 hh > sotv RNAi ( J and J ′). E-Cad and Mys expressions were detected by antibody staining, and Nrg-GFP and Vkg-GFP expressions were detected using GFP trap lines. A cross-section for each marker is shown below each 2D projection image. Blue dotted lines indicate locations of cross-sections. (Scale bars, 50 µm.)

    Techniques Used: Expressing, Immunostaining, Staining, Marker

    12) Product Images from "Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8"

    Article Title: Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2018.1446660

    CCL18 acts as a bridging molecule between GAGs on EVs and cellular CCR8 (a–c) Heparan sulphate (a), dermatan sulphate (b) and chondroitin sulphate (c) GAGs are present on EV membranes, as determined by ELISA. (d) EV uptake is prevented by heparin (25 µg/mL) and by (e) Heparinase III (H’se III) (2 miU every 2 h for 6 h at 37°C) treatment of EV isolates. *** indicates p-value ≤0.001 as determined by t-test. (f) Proposed model: GAGs present on the EV membrane bind CCL18 which connects with cellular CCR8 promoting EV uptake. Error bars represent SD of three independent experiments.
    Figure Legend Snippet: CCL18 acts as a bridging molecule between GAGs on EVs and cellular CCR8 (a–c) Heparan sulphate (a), dermatan sulphate (b) and chondroitin sulphate (c) GAGs are present on EV membranes, as determined by ELISA. (d) EV uptake is prevented by heparin (25 µg/mL) and by (e) Heparinase III (H’se III) (2 miU every 2 h for 6 h at 37°C) treatment of EV isolates. *** indicates p-value ≤0.001 as determined by t-test. (f) Proposed model: GAGs present on the EV membrane bind CCL18 which connects with cellular CCR8 promoting EV uptake. Error bars represent SD of three independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    13) Product Images from "Interaction of poly-l-lysine coating and heparan sulfate proteoglycan on magnetic nanoparticle uptake by tumor cells"

    Article Title: Interaction of poly-l-lysine coating and heparan sulfate proteoglycan on magnetic nanoparticle uptake by tumor cells

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S156029

    Heparin (Hep) attenuated the enhancing effects of ( A , C ) PLL and ( B , D ) PLL coating on internalization of ( A , C ) Dex-MNPs and ( B , D ) PLL-MNPs, respectively. Notes: A magnetic field was applied for 5 minutes (Mag–) or 3 hours (Mag+) during the 3-hour incubation of Hep (0.1–10 IU/mL) or sialic acid (SA; 100 μM) with ( A , B ) U87MG or ( C , D ) LN229 cells. In some experiments, U87MG cells were incubated with 5 mIU/mL of heparinase III (Hep III) for 3 hours prior to PLL-MNP + treatment ( A insert). Values are means ± SE (n=4). * ,◊ P
    Figure Legend Snippet: Heparin (Hep) attenuated the enhancing effects of ( A , C ) PLL and ( B , D ) PLL coating on internalization of ( A , C ) Dex-MNPs and ( B , D ) PLL-MNPs, respectively. Notes: A magnetic field was applied for 5 minutes (Mag–) or 3 hours (Mag+) during the 3-hour incubation of Hep (0.1–10 IU/mL) or sialic acid (SA; 100 μM) with ( A , B ) U87MG or ( C , D ) LN229 cells. In some experiments, U87MG cells were incubated with 5 mIU/mL of heparinase III (Hep III) for 3 hours prior to PLL-MNP + treatment ( A insert). Values are means ± SE (n=4). * ,◊ P

    Techniques Used: Incubation

    14) Product Images from "Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells"

    Article Title: Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-016-0073-8

    Pharmacological knockdown of HSPG mitigates Aβ 1-40 -induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM; panels a , b ) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c ), washed, and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ 1-40, human VSMC cells were treated with Aβ 1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d ). Results are representative of 3 independent experiments performed in triplicate. *p
    Figure Legend Snippet: Pharmacological knockdown of HSPG mitigates Aβ 1-40 -induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM; panels a , b ) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c ), washed, and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ 1-40, human VSMC cells were treated with Aβ 1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d ). Results are representative of 3 independent experiments performed in triplicate. *p

    Techniques Used: Concentration Assay, Fluorescence, Immunoprecipitation

    15) Product Images from "Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin"

    Article Title: Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.17.8722-8728.2002

    Enzymatic digestion of HS eliminates binding to FN and gene transfer in the absence of PB. Amphotropic (Ampho) or gp70-deficient (Phoenix-gp) Mo-MuLV was incubated with either heparinase III (5 mIU/ml) or chondroitinase ABC (0.1 U/ml) for 3 h at 37°C. Control samples were incubated at 37°C for the same length of time in the absence of the enzyme. (A) The virus was incubated in 96-well FN-coated plates for ∼12 h at 25°C, and binding was evaluated with an ELISA for the matrix protein p30. The OD was normalized to that of control virus. (B) NIH 3T3 target cells in 96-well FN-coated plates or TCP (5,000 cells/well) were incubated with the enzyme-treated virus in the presence or absence of PB. (C) NIH 3T3 cells were added to 96-well FN-coated plates or TCP at 5,000 cells/well. The next day, the target cells were exposed to either culture medium (control) or heparinase III (5 mIU/ml) for 3 h at 37°C. The cells were washed with PBS and exposed to untreated virus stock. (B and C) The transduction efficiencies were quantified by measurements of β-galactosidase(βGal) activity once the target cells reached confluence. Values are means ± standard deviations of duplicate samples in a representative experiment. Asterisks denote very small values (close to zero).
    Figure Legend Snippet: Enzymatic digestion of HS eliminates binding to FN and gene transfer in the absence of PB. Amphotropic (Ampho) or gp70-deficient (Phoenix-gp) Mo-MuLV was incubated with either heparinase III (5 mIU/ml) or chondroitinase ABC (0.1 U/ml) for 3 h at 37°C. Control samples were incubated at 37°C for the same length of time in the absence of the enzyme. (A) The virus was incubated in 96-well FN-coated plates for ∼12 h at 25°C, and binding was evaluated with an ELISA for the matrix protein p30. The OD was normalized to that of control virus. (B) NIH 3T3 target cells in 96-well FN-coated plates or TCP (5,000 cells/well) were incubated with the enzyme-treated virus in the presence or absence of PB. (C) NIH 3T3 cells were added to 96-well FN-coated plates or TCP at 5,000 cells/well. The next day, the target cells were exposed to either culture medium (control) or heparinase III (5 mIU/ml) for 3 h at 37°C. The cells were washed with PBS and exposed to untreated virus stock. (B and C) The transduction efficiencies were quantified by measurements of β-galactosidase(βGal) activity once the target cells reached confluence. Values are means ± standard deviations of duplicate samples in a representative experiment. Asterisks denote very small values (close to zero).

    Techniques Used: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Transduction, Activity Assay

    16) Product Images from "Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection"

    Article Title: Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00432-17

    Golgi compartment-resident TM9SF2-dependent localization and stability of NDST1. (A) The cell surface expressions of HS, syndecan-1, and glypican-3 in wild-type HAP1 and HAP1ΔTM9SF2 cells were analyzed by flow cytometry. The numbers indicate the G-MFIs. (B) Intracellular localization of endogenous TM9SF2. Cells were methanol fixed and double stained with anti-TM9SF2, anti-GM130, or anti-Golgin97 antibodies. Green, TM9SF2; red, GM130 or Golgin97 (Golgi apparatus markers). (C) The cell surface expression of TM9SF2 in wild-type HAP1 (middle) and mutant HAP1ΔTM9SF2 cells (right) was analyzed by flow cytometry. The numbers indicate the G-MFIs. Wild-type HAP1 cells stained only with secondary antibody were used as the negative control (left). (D) Molecular sizes of glypican-3 in various HAP1-derived knockout and rescued cells. Cell lysates of these cells were subjected to immunoblotting to analyze the expression of glypican-3. (E) Analysis of N-sulfation and HS chain numbers in wild-type HAP1 (wild type+vector), TM9SF2-knockout cells (ΔTM9SF2+vector), and rescued TM9SF2-knockout cells (ΔTM9SF2+TM9SF2). Cells were incubated with or without 0.3 U of heparinase III at 37°C for 1 h and then stained with F58-10E4 (10E4) to detect HS expression and F69-3G10 (3G10) to determine HS chain numbers. Cells stained only with secondary antibody (Alexa 488-conjugated anti-mouse Ig or IgM antibody) were used as the negative control (without first antibody). Solid line, no enzyme treatment; dotted line, heparinase III treatment. (F) Quantitative analysis of the results in panel E. Relative ratios of cell surface N-sulfated HS (left columns), HS chain numbers (middle), and N-sulfates per HS chain (right) were calculated from the G-MFI values detected in panel E. The data represent the means ± the SE for three independent experiments. * , P
    Figure Legend Snippet: Golgi compartment-resident TM9SF2-dependent localization and stability of NDST1. (A) The cell surface expressions of HS, syndecan-1, and glypican-3 in wild-type HAP1 and HAP1ΔTM9SF2 cells were analyzed by flow cytometry. The numbers indicate the G-MFIs. (B) Intracellular localization of endogenous TM9SF2. Cells were methanol fixed and double stained with anti-TM9SF2, anti-GM130, or anti-Golgin97 antibodies. Green, TM9SF2; red, GM130 or Golgin97 (Golgi apparatus markers). (C) The cell surface expression of TM9SF2 in wild-type HAP1 (middle) and mutant HAP1ΔTM9SF2 cells (right) was analyzed by flow cytometry. The numbers indicate the G-MFIs. Wild-type HAP1 cells stained only with secondary antibody were used as the negative control (left). (D) Molecular sizes of glypican-3 in various HAP1-derived knockout and rescued cells. Cell lysates of these cells were subjected to immunoblotting to analyze the expression of glypican-3. (E) Analysis of N-sulfation and HS chain numbers in wild-type HAP1 (wild type+vector), TM9SF2-knockout cells (ΔTM9SF2+vector), and rescued TM9SF2-knockout cells (ΔTM9SF2+TM9SF2). Cells were incubated with or without 0.3 U of heparinase III at 37°C for 1 h and then stained with F58-10E4 (10E4) to detect HS expression and F69-3G10 (3G10) to determine HS chain numbers. Cells stained only with secondary antibody (Alexa 488-conjugated anti-mouse Ig or IgM antibody) were used as the negative control (without first antibody). Solid line, no enzyme treatment; dotted line, heparinase III treatment. (F) Quantitative analysis of the results in panel E. Relative ratios of cell surface N-sulfated HS (left columns), HS chain numbers (middle), and N-sulfates per HS chain (right) were calculated from the G-MFI values detected in panel E. The data represent the means ± the SE for three independent experiments. * , P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Expressing, Mutagenesis, Negative Control, Derivative Assay, Knock-Out, Plasmid Preparation, Incubation

    17) Product Images from "Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells"

    Article Title: Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.23302

    Immunofluorescence staining for HA in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HA. Treatment with heparinase III did not change the intensity of HA fluorescence
    Figure Legend Snippet: Immunofluorescence staining for HA in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HA. Treatment with heparinase III did not change the intensity of HA fluorescence

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Fluorescence

    Immunofluorescence staining for HSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HSPG. Treatment with heparinase III significantly reduced expression of HSPG
    Figure Legend Snippet: Immunofluorescence staining for HSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain HSPG. Treatment with heparinase III significantly reduced expression of HSPG

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Expressing

    ESC-derived EC gene response to shear stress (5 dyn/cm 2 for 8h) after treatment with heparinase III (20 mU/ml). Relative gene expression (gene/GAPDH) was measured by qPCR and normalized to its own static control case prior to enzyme treatment. Heparinase
    Figure Legend Snippet: ESC-derived EC gene response to shear stress (5 dyn/cm 2 for 8h) after treatment with heparinase III (20 mU/ml). Relative gene expression (gene/GAPDH) was measured by qPCR and normalized to its own static control case prior to enzyme treatment. Heparinase

    Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

    Immunofluorescence staining for CSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain CSPG. Treatment with heparinase III did not change the intensity of CSPG fluorescence
    Figure Legend Snippet: Immunofluorescence staining for CSPG in ESC-derived ECs before and after treatment with heparinase III. (Top panels) show that the surfaces of untreated ESC-derived ECs contain CSPG. Treatment with heparinase III did not change the intensity of CSPG fluorescence

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Fluorescence

    18) Product Images from "Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation"

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000666

    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Figure Legend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Techniques Used: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    PrP C and PrP Sc immunoprecipitate with glypican-1. ( A ) SH-SY5Y cells expressing PrP C , ( B ) N2a cells or ( C and D ) ScN2a cells were lysed in the indicated detergents and then immunoprecipitated with a polyclonal glypican-1 antibody and where indicated, co-incubated with 50 µM heparin. Those samples pretreated with heparinase I and heparinase III were lysed with Triton X-100 followed by immunoprecipitation with glypican-1 antibody. In (D), immunopreciptiates from ScN2a cells were digested with PK. All immunoprecipitates were subsequently blotted for PrP. TX, Triton X-100; OG, octylglucoside; SK, sarkosyl.
    Figure Legend Snippet: PrP C and PrP Sc immunoprecipitate with glypican-1. ( A ) SH-SY5Y cells expressing PrP C , ( B ) N2a cells or ( C and D ) ScN2a cells were lysed in the indicated detergents and then immunoprecipitated with a polyclonal glypican-1 antibody and where indicated, co-incubated with 50 µM heparin. Those samples pretreated with heparinase I and heparinase III were lysed with Triton X-100 followed by immunoprecipitation with glypican-1 antibody. In (D), immunopreciptiates from ScN2a cells were digested with PK. All immunoprecipitates were subsequently blotted for PrP. TX, Triton X-100; OG, octylglucoside; SK, sarkosyl.

    Techniques Used: Expressing, Immunoprecipitation, Incubation

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P
    Figure Legend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Techniques Used: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    19) Product Images from "Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells *"

    Article Title: Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.028498

    Heparin competes with Wnt5A binding of HS, and removal of HS decreases motility in melanoma cell lines. Cells were treated with increasing doses of heparin, and the medium of the cells was examined for Wnt5A release. A , in the presence of heparin, Wnt5A accumulates in the medium. The addition of rWnt5A to cells in the absence of heparin results in its rapid uptake and internalization, but in the presence of heparin, rWnt5A just accumulates in the medium. B , PKC signaling is affected by high doses of heparin, regardless of whether rWnt5A is added. C , cells were treated with 2 milliunits/ml heparinase III for 24 h and examined for PKC signaling. PKC signaling is decreased upon heparinase III treatment and partially reconstituted upon the addition of rWnt5A. D , representative images of the motility assays using M93-047 cells demonstrating that heparinase III treatment decreased the rate of wound closure. E , graphical representation of UACC903 scratch closure rates demonstrating that rWnt5A addition can overcome heparinase III treatment. F , quantitative extracellular matrix invasion assays also demonstrate that heparinase III treatment decreases the rate of melanoma cell invasion, and this can be restored upon the addition of exogenous Wnt5A to the media of the cells ( n = 3; error bars show S.D.; **, p
    Figure Legend Snippet: Heparin competes with Wnt5A binding of HS, and removal of HS decreases motility in melanoma cell lines. Cells were treated with increasing doses of heparin, and the medium of the cells was examined for Wnt5A release. A , in the presence of heparin, Wnt5A accumulates in the medium. The addition of rWnt5A to cells in the absence of heparin results in its rapid uptake and internalization, but in the presence of heparin, rWnt5A just accumulates in the medium. B , PKC signaling is affected by high doses of heparin, regardless of whether rWnt5A is added. C , cells were treated with 2 milliunits/ml heparinase III for 24 h and examined for PKC signaling. PKC signaling is decreased upon heparinase III treatment and partially reconstituted upon the addition of rWnt5A. D , representative images of the motility assays using M93-047 cells demonstrating that heparinase III treatment decreased the rate of wound closure. E , graphical representation of UACC903 scratch closure rates demonstrating that rWnt5A addition can overcome heparinase III treatment. F , quantitative extracellular matrix invasion assays also demonstrate that heparinase III treatment decreases the rate of melanoma cell invasion, and this can be restored upon the addition of exogenous Wnt5A to the media of the cells ( n = 3; error bars show S.D.; **, p

    Techniques Used: Binding Assay

    20) Product Images from "Heparan Sulfate, Including that in Bruch's Membrane, Inhibits the Complement Alternative Pathway: Implications for Age-related Macular Degeneration"

    Article Title: Heparan Sulfate, Including that in Bruch's Membrane, Inhibits the Complement Alternative Pathway: Implications for Age-related Macular Degeneration

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0903596

    Electron micrograph of human Bruch’s membrane/choroid stained for proteoglycans using Cupromeronic Blue. ( A ) HS [arrowheads in inner collagenous layer (ICL)], chondroitin sulfate and dermatan sulfate are stained, and can be distinguished based on their length/diameter and locations (Call and Hollyfield, 1990). ( B ) The HS associated with the basal lamina of pigment epithelium and choriocapillaris has disappeared following heparinase III treatment. CEL, central elastin layer; OCL, outer collagen layer. Scale bar = 500 nm.
    Figure Legend Snippet: Electron micrograph of human Bruch’s membrane/choroid stained for proteoglycans using Cupromeronic Blue. ( A ) HS [arrowheads in inner collagenous layer (ICL)], chondroitin sulfate and dermatan sulfate are stained, and can be distinguished based on their length/diameter and locations (Call and Hollyfield, 1990). ( B ) The HS associated with the basal lamina of pigment epithelium and choriocapillaris has disappeared following heparinase III treatment. CEL, central elastin layer; OCL, outer collagen layer. Scale bar = 500 nm.

    Techniques Used: Staining

    21) Product Images from "Shear-induced endothelial cell-cell junction inclination"

    Article Title: Shear-induced endothelial cell-cell junction inclination

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00156.2010

    Glycocalyx degradation did not alter flow-induced EC-cell junction inclination. HUVECs were subjected to a 16 dyn/cm 2 steady shear stress for 30 min after 2-h pretreatment with 50 μg/ml heparinase III (Hep). A and B : PECAM-1 immunostaining with XZ cross sections showing inclination of the EC-cell junction in Hep-pretreated cells and control (Ctrl)-untreated experiment. Scale bars are 20 μm. C : removal of heparan sulfate by Hep treatment was confirmed by Western blot. D : EC-cell junction inclination angles for each condition described above.
    Figure Legend Snippet: Glycocalyx degradation did not alter flow-induced EC-cell junction inclination. HUVECs were subjected to a 16 dyn/cm 2 steady shear stress for 30 min after 2-h pretreatment with 50 μg/ml heparinase III (Hep). A and B : PECAM-1 immunostaining with XZ cross sections showing inclination of the EC-cell junction in Hep-pretreated cells and control (Ctrl)-untreated experiment. Scale bars are 20 μm. C : removal of heparan sulfate by Hep treatment was confirmed by Western blot. D : EC-cell junction inclination angles for each condition described above.

    Techniques Used: Flow Cytometry, Immunostaining, Western Blot

    22) Product Images from "Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody"

    Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14793-z

    HS and CS chains are responsible for endocytosis of 3D8 scFv. At 24 h post-transfection with plasmids encoding SDC2-GFP, HA-GPC3, CD44-GFP, or HA-BCAN, HeLa cells were treated with 500 mIU/ml heparinase III or 500 mIU/ml chondroitinase ABC for 2 h at 37 °C. Then, cells were treated for 6 h at 37 °C with 3D8 scFv, followed by immunoprecipitation with anti-GFP, anti-GPC3, anti-CD44, or anti-HA antibodies. Proteins in the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting.
    Figure Legend Snippet: HS and CS chains are responsible for endocytosis of 3D8 scFv. At 24 h post-transfection with plasmids encoding SDC2-GFP, HA-GPC3, CD44-GFP, or HA-BCAN, HeLa cells were treated with 500 mIU/ml heparinase III or 500 mIU/ml chondroitinase ABC for 2 h at 37 °C. Then, cells were treated for 6 h at 37 °C with 3D8 scFv, followed by immunoprecipitation with anti-GFP, anti-GPC3, anti-CD44, or anti-HA antibodies. Proteins in the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting.

    Techniques Used: Transfection, Immunoprecipitation

    3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.
    Figure Legend Snippet: 3D8 scFv binds to cell surface HSPGs and CSPGs. ( a ) Flow cytometry analysis of cell surface expression of endogenous HSPGs and CSPGs in HeLa cells. ( b ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs and CSPGs. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) and HW6 scFv (10 μM) (negative control). Thereafter, cells were incubated with a primary antibody mixture containing a rabbit anti-3D8 scFv antibody and a mouse IgM anti-HS antibody, or a rabbit anti-3D8 scFv antibody and a mouse IgM anti-CS antibody. After washing, cells were incubated with a secondary antibody mixture comprising TRITC-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgM. ( c ) Confocal microscopy to detect 3D8 scFv binding to cell surface HSPGs in the presence of soluble HS and CS chains. HeLa cells were incubated for 1 h at 4 °C with 3D8 scFv (10 μM) in the absence ( upper panel ) or presence ( middle or lower panel ) of heparin (10 μg/ml) or CS-A (10 μg/ml), followed by the procedures described in ( b ). ( d,e ) Flow cytometry ( d ) and confocal microscopy ( e ) to detect the cell surface sugar chains (HS and CS) and cell surface binding of 3D8 scFv to HeLa cells pre-treated with heparinase III (10 mIU/ml) and chondroitinase ABC (100 mIU/ml). ( b,c,e ) Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm.

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

    23) Product Images from "Internalized Tau Oligomers Cause Neurodegeneration by Inducing Accumulation of Pathogenic Tau in Human Neurons Derived from Induced Pluripotent Stem Cells"

    Article Title: Internalized Tau Oligomers Cause Neurodegeneration by Inducing Accumulation of Pathogenic Tau in Human Neurons Derived from Induced Pluripotent Stem Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1523-15.2015

    Internalization of tau seeds by hiPSC neurons. A , Representative live-cell images of Cy3-tau conjugate (red) show the internalization of tau monomers and oligomers, but not fibrils (10 d preparation), 1 h after addition. Hoechst was used to stain nuclei (blue). Scale bar, 50 μm. B , Images demonstrate the ability of background suppressor to efficiently suppress the fluorescence of Cy3-tau oligomers added to a well with a media, at the same concentration used in the uptake assay. C , Graph shows the decrease in the cellular intensity of Cy3-tau oligomers after heparinase III treatment. hiPSC neurons pretreated for 3 h with heparinase III were seeded with Cy3-tau oligomers for 1 h (Student's t test, *** p
    Figure Legend Snippet: Internalization of tau seeds by hiPSC neurons. A , Representative live-cell images of Cy3-tau conjugate (red) show the internalization of tau monomers and oligomers, but not fibrils (10 d preparation), 1 h after addition. Hoechst was used to stain nuclei (blue). Scale bar, 50 μm. B , Images demonstrate the ability of background suppressor to efficiently suppress the fluorescence of Cy3-tau oligomers added to a well with a media, at the same concentration used in the uptake assay. C , Graph shows the decrease in the cellular intensity of Cy3-tau oligomers after heparinase III treatment. hiPSC neurons pretreated for 3 h with heparinase III were seeded with Cy3-tau oligomers for 1 h (Student's t test, *** p

    Techniques Used: Staining, Fluorescence, Concentration Assay

    24) Product Images from "Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells"

    Article Title: Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-016-0073-8

    Pharmacological knockdown of HSPG mitigates Aβ 1-40 -induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM; panels a , b ) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c ), washed, and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ 1-40, human VSMC cells were treated with Aβ 1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d ). Results are representative of 3 independent experiments performed in triplicate. *p
    Figure Legend Snippet: Pharmacological knockdown of HSPG mitigates Aβ 1-40 -induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM; panels a , b ) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c ), washed, and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ 1-40, human VSMC cells were treated with Aβ 1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d ). Results are representative of 3 independent experiments performed in triplicate. *p

    Techniques Used: Concentration Assay, Fluorescence, Immunoprecipitation

    25) Product Images from "Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells"

    Article Title: Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-016-0073-8

    Pharmacological knockdown of HSPG mitigates Aβ 1-40 -induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM; panels a , b ) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c ), washed, and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ 1-40, human VSMC cells were treated with Aβ 1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d ). Results are representative of 3 independent experiments performed in triplicate. *p
    Figure Legend Snippet: Pharmacological knockdown of HSPG mitigates Aβ 1-40 -induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H 2 XRos (MTR; 5 μM; panels a , b ) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c ), washed, and treated with Aβ 1-40 . In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ 1-40, human VSMC cells were treated with Aβ 1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d ). Results are representative of 3 independent experiments performed in triplicate. *p

    Techniques Used: Concentration Assay, Fluorescence, Immunoprecipitation

    26) Product Images from "Peptide-Derivatized SB105-A10 Dendrimer Inhibits the Infectivity of R5 and X4 HIV-1 Strains in Primary PBMCs and Cervicovaginal Histocultures"

    Article Title: Peptide-Derivatized SB105-A10 Dendrimer Inhibits the Infectivity of R5 and X4 HIV-1 Strains in Primary PBMCs and Cervicovaginal Histocultures

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076482

    Flow cytometry analysis of binding between SB105-A10 and heparan sulphates. Typical experiments were shown. ( A ), Different concentrations of FITC-conjugated SB105-A10 (from 0 to 250 µg/ml) were assayed for 1 hour at 4°C on activated PBMCs (5×10 6 /ml). The saturation was achieved at 25 µg/ml. ( B ), Activated PBMCs (5×10 6 /ml) were treated with 25 µg/ml of FITC-conjugated SB105-A10 for 1 hour at 4°C and then washed with PBS containing 2 M NaCl, a treatment known to remove cationic polypeptides from cell surface HSPGs. ( C ), Activated PBMCs (5×10 6 /ml) were incubated with heparinase III for 2 h at 37°C or left untreated before the binding assay with 25 µg/ml of FITC-conjugated SB105-A10. Both heparinase III and 2 M NaCl treatments reduce but not abolish the specific fluorescence signal suggesting an interaction between SB105-A10 and cell membrane that is not related to HSPGs.
    Figure Legend Snippet: Flow cytometry analysis of binding between SB105-A10 and heparan sulphates. Typical experiments were shown. ( A ), Different concentrations of FITC-conjugated SB105-A10 (from 0 to 250 µg/ml) were assayed for 1 hour at 4°C on activated PBMCs (5×10 6 /ml). The saturation was achieved at 25 µg/ml. ( B ), Activated PBMCs (5×10 6 /ml) were treated with 25 µg/ml of FITC-conjugated SB105-A10 for 1 hour at 4°C and then washed with PBS containing 2 M NaCl, a treatment known to remove cationic polypeptides from cell surface HSPGs. ( C ), Activated PBMCs (5×10 6 /ml) were incubated with heparinase III for 2 h at 37°C or left untreated before the binding assay with 25 µg/ml of FITC-conjugated SB105-A10. Both heparinase III and 2 M NaCl treatments reduce but not abolish the specific fluorescence signal suggesting an interaction between SB105-A10 and cell membrane that is not related to HSPGs.

    Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Incubation, Fluorescence

    27) Product Images from "Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells *"

    Article Title: Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.142703

    Glycosaminoglycans inhibit Factor H-mediated pneumococcal adhesion to human lung epithelial cells. A , adherence of pneumococci via Factor H to lung epithelial A549 cells in the absence ( control ) or the presence of heparin (50 units/ml), dermatan sulfate (100 μg/ml), or after pretreatment with heparinase III (10 milliunits/ml) was estimated by quantifying the colony-forming units ( cfu ) per well obtained from plating onto blood agar plates. The infection assays were conducted with or without ( none ) pretreatment of pneumococci with Factor H. *, p
    Figure Legend Snippet: Glycosaminoglycans inhibit Factor H-mediated pneumococcal adhesion to human lung epithelial cells. A , adherence of pneumococci via Factor H to lung epithelial A549 cells in the absence ( control ) or the presence of heparin (50 units/ml), dermatan sulfate (100 μg/ml), or after pretreatment with heparinase III (10 milliunits/ml) was estimated by quantifying the colony-forming units ( cfu ) per well obtained from plating onto blood agar plates. The infection assays were conducted with or without ( none ) pretreatment of pneumococci with Factor H. *, p

    Techniques Used: Infection

    28) Product Images from "Syndecan-4 mediates macrophage uptake of group V secretory phospholipase A2-modified LDL"

    Article Title: Syndecan-4 mediates macrophage uptake of group V secretory phospholipase A2-modified LDL

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M800450-JLR200

    Macrophage uptake of group V sPLA2-modified LDL (GV-LDL) depends on cellular proteoglycans. J-774 cells were preincubated for 4 h in the presence or absence of 1 U/ml heparinase III + 0.15 U/ml chondroitinase ABC in PBS containing 0.1 M sodium acetate
    Figure Legend Snippet: Macrophage uptake of group V sPLA2-modified LDL (GV-LDL) depends on cellular proteoglycans. J-774 cells were preincubated for 4 h in the presence or absence of 1 U/ml heparinase III + 0.15 U/ml chondroitinase ABC in PBS containing 0.1 M sodium acetate

    Techniques Used: Modification

    29) Product Images from "Adaptation of Sindbis Virus to BHK Cells Selects for Use of Heparan Sulfate as an Attachment Receptor"

    Article Title: Adaptation of Sindbis Virus to BHK Cells Selects for Use of Heparan Sulfate as an Attachment Receptor

    Journal: Journal of Virology

    doi:

    Effect of heparinase digestion on plaque-forming efficiency of TR339, TRSB, and TRSB-R114 on BHK cells. (A) BHK cell monolayers were digested with increasing concentrations of heparinase I, followed by three rounds of washing with virus buffer and infection with 100 to 200 PFU of TR339 (squares), TRSB (circles), or TRSB-R114 (triangles). (B) BHK cell monolayers were digested with increasing concentrations of heparinase III, washed, and infected as above. Data are averages of two or three assays at each concentration.
    Figure Legend Snippet: Effect of heparinase digestion on plaque-forming efficiency of TR339, TRSB, and TRSB-R114 on BHK cells. (A) BHK cell monolayers were digested with increasing concentrations of heparinase I, followed by three rounds of washing with virus buffer and infection with 100 to 200 PFU of TR339 (squares), TRSB (circles), or TRSB-R114 (triangles). (B) BHK cell monolayers were digested with increasing concentrations of heparinase III, washed, and infected as above. Data are averages of two or three assays at each concentration.

    Techniques Used: Infection, Concentration Assay

    Effect of heparinase digestion on binding of TRSB (circles) and TRSB-R114 (triangles) to BHK cells. (A) BHK cell monolayers were digested with increasing concentrations of heparinase I, followed by three rounds of washing with VB, suspension, and use in binding assays (5 × 10 4 cpm of radiolabeled virus per reaction). (B) BHK cell monolayers were digested with increasing concentrations of heparinase III and processed as above. Data are averages of two or three binding reactions at each concentration. In these assays, binding of TR339 to digested or undigested cells was not above background cpm measured in cell-free control reactions.
    Figure Legend Snippet: Effect of heparinase digestion on binding of TRSB (circles) and TRSB-R114 (triangles) to BHK cells. (A) BHK cell monolayers were digested with increasing concentrations of heparinase I, followed by three rounds of washing with VB, suspension, and use in binding assays (5 × 10 4 cpm of radiolabeled virus per reaction). (B) BHK cell monolayers were digested with increasing concentrations of heparinase III and processed as above. Data are averages of two or three binding reactions at each concentration. In these assays, binding of TR339 to digested or undigested cells was not above background cpm measured in cell-free control reactions.

    Techniques Used: Binding Assay, Concentration Assay

    30) Product Images from "Repellent Guidance of Regenerating Optic Axons by Chondroitin Sulfate Glycosaminoglycans in Zebrafish"

    Article Title: Repellent Guidance of Regenerating Optic Axons by Chondroitin Sulfate Glycosaminoglycans in Zebrafish

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-03-00842.2002

    Outlines of the areas invaded by regenerating optic fibers in sections of the magnocellular superficial/posterior pretectal nucleus after different treatments. Chartings of all cases that received chondroitinase ( A ), vehicle ( B ), no injection ( C ), or heparinase injection ( D ) after contralateral optic nerve crush are shown; dorsal is at the top ; lateral is left . The magnocellular superficial/posterior pretectal nucleus stretches over two to three cross sections. These are depicted in columns for the individual cases. All chartings are organized as in the first case in B , with the most rostral section on the bottom and the most caudal section on the top ( R → C ). The parvocellular superficial ( PSp ), magnocellular superficial ( PSm ), accessory ( APN ), central ( CPN ), and posterior ( PO ) pretectal nuclei, as well as the dorsal accessory optic nucleus ( DAO ), are outlined as indicated for the first case in B . The area taken by fibers invading the magnocellular superficial/posterior pretectal nucleus in cross sections is black . Fibers reinnervating their regular terminal fields in the dorsal accessory optic nucleus and the central pretectal nucleus are gray . Fibers reinnervating the parvocellular superficial pretectal nucleus after a lesion have been omitted for clarity. Animals were scored as having fibers invading the magnocellular superficial/posterior pretectal nucleus when fibers were present in these nuclei in at least two consecutive sections (see Materials and Methods). Cases are sorted accordingly (+, invasion of fibers; −, no invasion of fibers), and the percentages of cases with fibers in the magnocellular superficial/posterior pretectal nucleus are given. The proportion of cases with fibers in the magnocellular superficial/posterior pretectal nucleus after chondroitinase treatment is highly significantly increased ( p = 0.003) compared with vehicle-injected and uninjected controls. *Note that the heparinase preparation contained chondroitinase activity (see Results). Scale bar, 200 μm.
    Figure Legend Snippet: Outlines of the areas invaded by regenerating optic fibers in sections of the magnocellular superficial/posterior pretectal nucleus after different treatments. Chartings of all cases that received chondroitinase ( A ), vehicle ( B ), no injection ( C ), or heparinase injection ( D ) after contralateral optic nerve crush are shown; dorsal is at the top ; lateral is left . The magnocellular superficial/posterior pretectal nucleus stretches over two to three cross sections. These are depicted in columns for the individual cases. All chartings are organized as in the first case in B , with the most rostral section on the bottom and the most caudal section on the top ( R → C ). The parvocellular superficial ( PSp ), magnocellular superficial ( PSm ), accessory ( APN ), central ( CPN ), and posterior ( PO ) pretectal nuclei, as well as the dorsal accessory optic nucleus ( DAO ), are outlined as indicated for the first case in B . The area taken by fibers invading the magnocellular superficial/posterior pretectal nucleus in cross sections is black . Fibers reinnervating their regular terminal fields in the dorsal accessory optic nucleus and the central pretectal nucleus are gray . Fibers reinnervating the parvocellular superficial pretectal nucleus after a lesion have been omitted for clarity. Animals were scored as having fibers invading the magnocellular superficial/posterior pretectal nucleus when fibers were present in these nuclei in at least two consecutive sections (see Materials and Methods). Cases are sorted accordingly (+, invasion of fibers; −, no invasion of fibers), and the percentages of cases with fibers in the magnocellular superficial/posterior pretectal nucleus are given. The proportion of cases with fibers in the magnocellular superficial/posterior pretectal nucleus after chondroitinase treatment is highly significantly increased ( p = 0.003) compared with vehicle-injected and uninjected controls. *Note that the heparinase preparation contained chondroitinase activity (see Results). Scale bar, 200 μm.

    Techniques Used: Injection, Activity Assay

    31) Product Images from "Role of Glycocalyx in Flow-Induced Production of Nitric Oxide and Reactive Oxygen Species"

    Article Title: Role of Glycocalyx in Flow-Induced Production of Nitric Oxide and Reactive Oxygen Species

    Journal:

    doi: 10.1016/j.freeradbiomed.2009.05.034

    A) The area fraction of DHE fluorescent staining which indicates superoxides production in porcine superficial femoral artery after heparinase III, hyaluronidase, and neuraminidase treatment. Control (open bar) is shown as 100%. The data represent mean
    Figure Legend Snippet: A) The area fraction of DHE fluorescent staining which indicates superoxides production in porcine superficial femoral artery after heparinase III, hyaluronidase, and neuraminidase treatment. Control (open bar) is shown as 100%. The data represent mean

    Techniques Used: Staining

    A) Nitrite production rate in porcine superficial femoral artery after heparinase III treatment. Control (open bar), after treatment with 15 mU/ml heparinase III (hatched bar; P
    Figure Legend Snippet: A) Nitrite production rate in porcine superficial femoral artery after heparinase III treatment. Control (open bar), after treatment with 15 mU/ml heparinase III (hatched bar; P

    Techniques Used:

    32) Product Images from "A New Monoclonal Antibody, mAb 4A12, Identifies a Role for the Glycosaminoglycan (GAG) Binding Domain of RANTES in the Antiviral Effect against HIV-1 and Intracellular Ca2+ Signaling "

    Article Title: A New Monoclonal Antibody, mAb 4A12, Identifies a Role for the Glycosaminoglycan (GAG) Binding Domain of RANTES in the Antiviral Effect against HIV-1 and Intracellular Ca2+ Signaling

    Journal: The Journal of Experimental Medicine

    doi:

    Glycanase treatment blocks RANTES binding. PBMCs were treated a mixture of glycanases consisting of 1 U/ml each heparinase II, heparinase III, and chondroitinase ABC or medium alone. Cells were stained for RANTES binding as detected with 4A12 or CCR5 expression as detected with 2D7. Fluorescence intensity was analyzed by flow cytometry. A and C represent RANTES binding ( thick line ) as compared with background ( thin line ) in untreated and glycanase treated cells, respectively. B and D represent 2D7 anti-CCR5 ( thick line ) staining as compared with isotype control ( thin line ) in untreated and glycanase-treated cells, respectively.
    Figure Legend Snippet: Glycanase treatment blocks RANTES binding. PBMCs were treated a mixture of glycanases consisting of 1 U/ml each heparinase II, heparinase III, and chondroitinase ABC or medium alone. Cells were stained for RANTES binding as detected with 4A12 or CCR5 expression as detected with 2D7. Fluorescence intensity was analyzed by flow cytometry. A and C represent RANTES binding ( thick line ) as compared with background ( thin line ) in untreated and glycanase treated cells, respectively. B and D represent 2D7 anti-CCR5 ( thick line ) staining as compared with isotype control ( thin line ) in untreated and glycanase-treated cells, respectively.

    Techniques Used: Binding Assay, Staining, Expressing, Fluorescence, Flow Cytometry, Cytometry

    Cell surface GAGs are required for RANTES to elicit intracellular Ca 2+ signals. Primary human lymphocytes activated with PHA for 3 d followed by expansion in IL-2 for 9 d were either treated with a mixture of glycanases consisting of 1 U/ml each of heparinase II, heparinase III, and chondroitinase ABC ( thin lines ) or medium alone ( thick lines ), as described in Materials and Methods. The cells were loaded with the Ca 2+ indicator dye Fluo-3, and intracellular Ca 2+ responses were elicited by either 3 nM RANTES ( A ) or 66 nM anti-CD3 (clone UCHT-1; B ) and recorded by flow cytometry as previously described ( 34 , 35 ).
    Figure Legend Snippet: Cell surface GAGs are required for RANTES to elicit intracellular Ca 2+ signals. Primary human lymphocytes activated with PHA for 3 d followed by expansion in IL-2 for 9 d were either treated with a mixture of glycanases consisting of 1 U/ml each of heparinase II, heparinase III, and chondroitinase ABC ( thin lines ) or medium alone ( thick lines ), as described in Materials and Methods. The cells were loaded with the Ca 2+ indicator dye Fluo-3, and intracellular Ca 2+ responses were elicited by either 3 nM RANTES ( A ) or 66 nM anti-CD3 (clone UCHT-1; B ) and recorded by flow cytometry as previously described ( 34 , 35 ).

    Techniques Used: Flow Cytometry, Cytometry

    33) Product Images from "Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells"

    Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgu164

    GEP interacts with HS on cell surface of liver cancer cell line. ( A – C ) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. ( D ) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.
    Figure Legend Snippet: GEP interacts with HS on cell surface of liver cancer cell line. ( A – C ) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. ( D ) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.

    Techniques Used: Incubation, Binding Assay, Expressing, Blocking Assay

    Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. ( A ) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. ( B and C ) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W 1 and W 2 ). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. ( D ) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). ( E ) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. ( F ) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.
    Figure Legend Snippet: Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. ( A ) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. ( B and C ) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W 1 and W 2 ). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. ( D ) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). ( E ) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. ( F ) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.

    Techniques Used: Binding Assay, Chromatography, Transfection, Incubation, Flow Cytometry, Recombinant, Purification, SDS Page, Staining, Western Blot

    34) Product Images from "Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells"

    Article Title: Filoviruses Utilize Glycosaminoglycans for Their Attachment to Target Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01621-12

    HSPGs mediate attachment of filovirus to the cells. 293T, Huh7, and HUVEC cells were pretreated or mock treated with different amounts of heparinase I or heparinase III and exposed to concentrated HIV pseudotypes bearing EBOV, MARV, or Lassa virus envelope
    Figure Legend Snippet: HSPGs mediate attachment of filovirus to the cells. 293T, Huh7, and HUVEC cells were pretreated or mock treated with different amounts of heparinase I or heparinase III and exposed to concentrated HIV pseudotypes bearing EBOV, MARV, or Lassa virus envelope

    Techniques Used:

    35) Product Images from "Serotype-Specific Entry of Dengue Virus into Liver Cells: Identification of the 37-Kilodalton/67-Kilodalton High-Affinity Laminin Receptor as a Dengue Virus Serotype 1 Receptor"

    Article Title: Serotype-Specific Entry of Dengue Virus into Liver Cells: Identification of the 37-Kilodalton/67-Kilodalton High-Affinity Laminin Receptor as a Dengue Virus Serotype 1 Receptor

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.22.12647-12656.2004

    Effects of enzyme and antibody-enzyme pretreatment on infection of HepG2 cells by dengue virus serotype 1. HepG2 cells were either untreated (controls) or treated with trypsin or a combination of trypsin and heparinase III or preincubated with an antibody against the 37/67-kDa high-affinity laminin receptor or treated with heparinase III and subsequently preincubated with an antibody against the 37/67-kDa high-affinity laminin receptor prior to infection with dengue virus serotype 1 at a MOI of 1. Levels of virus in the medium were assayed either immediately after the period of viral inoculation (unbound virus) or after 1.5 virus replication cycles (extracellular virus). Each experimental point is the sum of triplicate experiments with duplicate assay of titer. Error bars represent standard errors of the mean. Asterisks indicate a significant difference from the control (one-sample t test); a, P = 0.0030; b, P = 0.0024; c, P = 0.0006, d, P
    Figure Legend Snippet: Effects of enzyme and antibody-enzyme pretreatment on infection of HepG2 cells by dengue virus serotype 1. HepG2 cells were either untreated (controls) or treated with trypsin or a combination of trypsin and heparinase III or preincubated with an antibody against the 37/67-kDa high-affinity laminin receptor or treated with heparinase III and subsequently preincubated with an antibody against the 37/67-kDa high-affinity laminin receptor prior to infection with dengue virus serotype 1 at a MOI of 1. Levels of virus in the medium were assayed either immediately after the period of viral inoculation (unbound virus) or after 1.5 virus replication cycles (extracellular virus). Each experimental point is the sum of triplicate experiments with duplicate assay of titer. Error bars represent standard errors of the mean. Asterisks indicate a significant difference from the control (one-sample t test); a, P = 0.0030; b, P = 0.0024; c, P = 0.0006, d, P

    Techniques Used: Infection

    36) Product Images from "Productive Dengue Virus Infection of Human Endothelial Cells Is Directed by Heparan Sulfate-Containing Proteoglycan Receptors ▿"

    Article Title: Productive Dengue Virus Infection of Human Endothelial Cells Is Directed by Heparan Sulfate-Containing Proteoglycan Receptors ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05008-11

    Enzymatic treatment of human endothelial cells. Endothelial cell monolayers were incubated with heparinase III (0.5 to 3 U/ml) (white bars) or chondroitinase (0.5 to 3 U/ml) (black bars) (A) or proteinase K (0.01 to 10 μg/ml, striped bars) (B)
    Figure Legend Snippet: Enzymatic treatment of human endothelial cells. Endothelial cell monolayers were incubated with heparinase III (0.5 to 3 U/ml) (white bars) or chondroitinase (0.5 to 3 U/ml) (black bars) (A) or proteinase K (0.01 to 10 μg/ml, striped bars) (B)

    Techniques Used: Incubation

    37) Product Images from "The recovery time course of the endothelial-cell glycocalyx in vivo and its implications in vitro"

    Article Title: The recovery time course of the endothelial-cell glycocalyx in vivo and its implications in vitro

    Journal: Circulation research

    doi: 10.1161/CIRCRESAHA.108.191585

    Systemic WBC count measured in 20 WT mice before (Day 0) and after administration with either either 100 µL saline, 100 U hyaluronidase, 1U heparinase III, or 0.2 µg of TNF-α. Each substance was administered to 5 WT mice, and each
    Figure Legend Snippet: Systemic WBC count measured in 20 WT mice before (Day 0) and after administration with either either 100 µL saline, 100 U hyaluronidase, 1U heparinase III, or 0.2 µg of TNF-α. Each substance was administered to 5 WT mice, and each

    Techniques Used: Mouse Assay

    38) Product Images from "Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors"

    Article Title: Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058340

    The obstruction or removal of negatively charged cellular interaction sites inhibit the HDV infection. A ) HepaRG cells were pre-incubated with increasing concentrations of poly-L-lysine for 30 min at 37°C and subsequently infected in presence of the polycation for 16 h at 37°C. B ) HDV was pre-incubated for 1 h at 37°C with heparin (100 µg/ml and 500 µg/ml), suramin (100 µg/ml) and dextran sulfate (100 µg/ml). Unbound polyanions were removed by ultrafiltration. HepaRG cells were incubated with the pre-treated viruses for 8 h at 37°C. C ) HepaRG cells were pre-incubated with heparin, suramin, dextran sulfate, pentosan polysulfate or poly-L-lysine for 1 h at 37°C. Cells were washed and incubated with HDV for 8 h at 37°C in absence of the compounds. D ) Sulfation of cellular proteoglycans was inhibited by treatment of HepaRG cells with increasing concentrations of sodium chlorate for 48 h prior to HDV infection. E ) HepaRG cells were incubated for 1 h at 37°C with the indicated concentrations of heparinase III or 1 µM preS/2-48 myr . The cells were washed and inoculated with HDV for 16 h at 37°C in presence of the substances. HDV-specific IF was performed for all experiments on day 6 p.i. Nuclei were stained with DAPI. The number of HDAg-positive cells and the total cell number were determined. The results are depicted as percentage of infected cells in comparison to the untreated control. In total, 822302 cells were counted for the analysis.
    Figure Legend Snippet: The obstruction or removal of negatively charged cellular interaction sites inhibit the HDV infection. A ) HepaRG cells were pre-incubated with increasing concentrations of poly-L-lysine for 30 min at 37°C and subsequently infected in presence of the polycation for 16 h at 37°C. B ) HDV was pre-incubated for 1 h at 37°C with heparin (100 µg/ml and 500 µg/ml), suramin (100 µg/ml) and dextran sulfate (100 µg/ml). Unbound polyanions were removed by ultrafiltration. HepaRG cells were incubated with the pre-treated viruses for 8 h at 37°C. C ) HepaRG cells were pre-incubated with heparin, suramin, dextran sulfate, pentosan polysulfate or poly-L-lysine for 1 h at 37°C. Cells were washed and incubated with HDV for 8 h at 37°C in absence of the compounds. D ) Sulfation of cellular proteoglycans was inhibited by treatment of HepaRG cells with increasing concentrations of sodium chlorate for 48 h prior to HDV infection. E ) HepaRG cells were incubated for 1 h at 37°C with the indicated concentrations of heparinase III or 1 µM preS/2-48 myr . The cells were washed and inoculated with HDV for 16 h at 37°C in presence of the substances. HDV-specific IF was performed for all experiments on day 6 p.i. Nuclei were stained with DAPI. The number of HDAg-positive cells and the total cell number were determined. The results are depicted as percentage of infected cells in comparison to the untreated control. In total, 822302 cells were counted for the analysis.

    Techniques Used: Infection, Incubation, Staining

    39) Product Images from "The signalling receptor MCAM coordinates apical-basal polarity and planar cell polarity during morphogenesis"

    Article Title: The signalling receptor MCAM coordinates apical-basal polarity and planar cell polarity during morphogenesis

    Journal: Nature Communications

    doi: 10.1038/ncomms15279

    MCAM/FGF4-dependent apical surface biogenesis. ( a ) Upper panel, cartoon of MCAM-BD 1–3. Lower panel, yeast zygotes obtained after mating the bait strain containing pGBKT7-MCAM with the library strain containing pGADT7-FGF2, FGF4, and FGF8. BD, DNA-binding domain; AD, activation domain; +, positive control with p53-BD; and −, negative control with empty AD vector and BD-lambda. ( b ) Co-immunoprecipitation of MCAM/FGF4 and FGFR1/FGF4 with the protein lysate treated with or without the heparinase I and heparinase III (0.06 IU ml −l ). ( c ) Kinetic dissociation constant ( K D ) of FGF4/MCAM, FGF4/FGFR1, or FGF4/ FGFR2 complexes was measured using a surface plasmon resonance method. ( d ) Distribution of polarized MCAM and unpolarized FGFR1 on chemotaxing cells. The source concentration of FGF4 in the chemotaxis assay is 10 ng ml −l . ( e ) Co-localization of endogenous MCAM and the apical marker aPKCζ in chemotaxing cells. Scale bar, 20 μm. ( f , g ) Time-lapse live-cells imaging of endogenous MCAM ( f ) or exogenous MCAM-RFP ( g ) at the leading edge of chemotaxing cells.
    Figure Legend Snippet: MCAM/FGF4-dependent apical surface biogenesis. ( a ) Upper panel, cartoon of MCAM-BD 1–3. Lower panel, yeast zygotes obtained after mating the bait strain containing pGBKT7-MCAM with the library strain containing pGADT7-FGF2, FGF4, and FGF8. BD, DNA-binding domain; AD, activation domain; +, positive control with p53-BD; and −, negative control with empty AD vector and BD-lambda. ( b ) Co-immunoprecipitation of MCAM/FGF4 and FGFR1/FGF4 with the protein lysate treated with or without the heparinase I and heparinase III (0.06 IU ml −l ). ( c ) Kinetic dissociation constant ( K D ) of FGF4/MCAM, FGF4/FGFR1, or FGF4/ FGFR2 complexes was measured using a surface plasmon resonance method. ( d ) Distribution of polarized MCAM and unpolarized FGFR1 on chemotaxing cells. The source concentration of FGF4 in the chemotaxis assay is 10 ng ml −l . ( e ) Co-localization of endogenous MCAM and the apical marker aPKCζ in chemotaxing cells. Scale bar, 20 μm. ( f , g ) Time-lapse live-cells imaging of endogenous MCAM ( f ) or exogenous MCAM-RFP ( g ) at the leading edge of chemotaxing cells.

    Techniques Used: Binding Assay, Activation Assay, Positive Control, Negative Control, Plasmid Preparation, Immunoprecipitation, SPR Assay, Concentration Assay, Chemotaxis Assay, Marker, Imaging

    40) Product Images from "The Signal Peptide of Staphylococcus aureus Panton Valentine Leukocidin LukS Component Mediates Increased Adhesion to Heparan Sulfates"

    Article Title: The Signal Peptide of Staphylococcus aureus Panton Valentine Leukocidin LukS Component Mediates Increased Adhesion to Heparan Sulfates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005042

    Inhibition of adhesion to collagen IV by heparinase. Isogenic strains of Staphylococcus aureus examined for their binding capacity (Y-axis, absorbance reading at 540 nm) to collagen IV (Sigma) coated on 96-well plates, at concentrations of 5 µg/mL. Heparinase III was added at increasing concentrations (X-axis) to collagen IV (Sigma) before the adhesion assay. Parental (LUG960) indicates the genetic background in which the genetic modifications were made; LukS-PV SP, parental carrying a plasmid encoding the LukS-PV signal peptide. The vertical lines indicate the standard deviations.
    Figure Legend Snippet: Inhibition of adhesion to collagen IV by heparinase. Isogenic strains of Staphylococcus aureus examined for their binding capacity (Y-axis, absorbance reading at 540 nm) to collagen IV (Sigma) coated on 96-well plates, at concentrations of 5 µg/mL. Heparinase III was added at increasing concentrations (X-axis) to collagen IV (Sigma) before the adhesion assay. Parental (LUG960) indicates the genetic background in which the genetic modifications were made; LukS-PV SP, parental carrying a plasmid encoding the LukS-PV signal peptide. The vertical lines indicate the standard deviations.

    Techniques Used: Inhibition, Binding Assay, Cell Adhesion Assay, Plasmid Preparation

    Related Articles

    Centrifugation:

    Article Title: Virion-associated viral fibroblast growth factor stimulates cell motility
    Article Snippet: .. To remove virion-bound vFGF, 3 × 107 AcBAC-HSP70vFGFHA virions in 3 ml of buffer (20 mM Tris-HCl, pH 7, 0.1mg/ml BSA, and 4 mM CaCl2 ) were treated with 1IU of heparinase III from Flavobacterium heparinum (Sigma Aldrich) at 27 °C for 4 h. After treatment, virions were purified by centrifugation at 24,000 × g through a 25% Nycoprep cushion. ..

    Immunostaining:

    Article Title: Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells
    Article Snippet: .. To cleave the cell surface HS prior to immunostaining, ESC-derived ECs were treated with Heparinase III (60mU/ml, Sigma chemical) and incubated at 37°C for 1h. ..

    Purification:

    Article Title: Virion-associated viral fibroblast growth factor stimulates cell motility
    Article Snippet: .. To remove virion-bound vFGF, 3 × 107 AcBAC-HSP70vFGFHA virions in 3 ml of buffer (20 mM Tris-HCl, pH 7, 0.1mg/ml BSA, and 4 mM CaCl2 ) were treated with 1IU of heparinase III from Flavobacterium heparinum (Sigma Aldrich) at 27 °C for 4 h. After treatment, virions were purified by centrifugation at 24,000 × g through a 25% Nycoprep cushion. ..

    Incubation:

    Article Title: Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum
    Article Snippet: .. Human bloodgroup O Rh+ erythrocytes (5% suspension in RPMI) were, before C-FDA labeling , incubated for 60–90 min with either heparinase III (25°C, pH 7.5; Sigma Chemical Co. ), chondroitinase ABC (37°C, pH 8.0; Sigma Chemical Co. ) or with clostridium perfringens neuraminidase (37°C, pH 6.0; Sigma Chemical Co. ). ..

    Article Title: Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells
    Article Snippet: .. To cleave the cell surface HS prior to immunostaining, ESC-derived ECs were treated with Heparinase III (60mU/ml, Sigma chemical) and incubated at 37°C for 1h. ..

    Activity Assay:

    Article Title: A Biomechanical Role for Perlecan in the Pericellular Matrix of Articular Cartilage
    Article Snippet: .. Heparinase III (heparitinase I; EC 4.2.2.8; Sigma-Aldrich, Inc., St. Louis, MO) is the most specific heparinase for HS, demonstrating no activity for heparin ( ). .. Cartilage sections were incubated in 50 μL of 6 U/mL (0.01 IU/mL) heparinase III solution in 20 mM Tris-HCl containing 0.1 mg/mL bovine serum albumin (BSA) and 4 mM CaCl2 , pH 7.0 at 37°C for 30 minutes.

    Labeling:

    Article Title: Identification of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) as the Rosetting Ligand of the Malaria Parasite P. falciparum
    Article Snippet: .. Human bloodgroup O Rh+ erythrocytes (5% suspension in RPMI) were, before C-FDA labeling , incubated for 60–90 min with either heparinase III (25°C, pH 7.5; Sigma Chemical Co. ), chondroitinase ABC (37°C, pH 8.0; Sigma Chemical Co. ) or with clostridium perfringens neuraminidase (37°C, pH 6.0; Sigma Chemical Co. ). ..

    Binding Assay:

    Article Title: Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction
    Article Snippet: .. In static controls, a fresh working solution of heparinase III was changed every 24 h. To rescue the ligand-receptor binding function of HSPG, we added 1 μg/ml heparin (H3149; Sigma) to 20 mM NaClO3 . ..

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    Millipore heparitinase
    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of <t>heparitinase</t> treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P
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    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of heparitinase treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P

    Journal: JCI Insight

    Article Title: Osteoblastic heparan sulfate regulates osteoprotegerin function and bone mass

    doi: 10.1172/jci.insight.89624

    Figure Lengend Snippet: Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of heparitinase treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P

    Article Snippet: Digestion of cell surface HS was performed by incubating live cells with 5 mIU/ml heparitinase (heparinase III) (MilliporeSigma, H8891) for 2 hours at 37°C.

    Techniques: Functional Assay, Generated, Affinity Chromatography, Binding Assay, Labeling, Staining, Marker, Activity Assay, Inhibition

    Heparin competes with Wnt5A binding of HS, and removal of HS decreases motility in melanoma cell lines. Cells were treated with increasing doses of heparin, and the medium of the cells was examined for Wnt5A release. A , in the presence of heparin, Wnt5A accumulates in the medium. The addition of rWnt5A to cells in the absence of heparin results in its rapid uptake and internalization, but in the presence of heparin, rWnt5A just accumulates in the medium. B , PKC signaling is affected by high doses of heparin, regardless of whether rWnt5A is added. C , cells were treated with 2 milliunits/ml heparinase III for 24 h and examined for PKC signaling. PKC signaling is decreased upon heparinase III treatment and partially reconstituted upon the addition of rWnt5A. D , representative images of the motility assays using M93-047 cells demonstrating that heparinase III treatment decreased the rate of wound closure. E , graphical representation of UACC903 scratch closure rates demonstrating that rWnt5A addition can overcome heparinase III treatment. F , quantitative extracellular matrix invasion assays also demonstrate that heparinase III treatment decreases the rate of melanoma cell invasion, and this can be restored upon the addition of exogenous Wnt5A to the media of the cells ( n = 3; error bars show S.D.; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells *

    doi: 10.1074/jbc.M109.028498

    Figure Lengend Snippet: Heparin competes with Wnt5A binding of HS, and removal of HS decreases motility in melanoma cell lines. Cells were treated with increasing doses of heparin, and the medium of the cells was examined for Wnt5A release. A , in the presence of heparin, Wnt5A accumulates in the medium. The addition of rWnt5A to cells in the absence of heparin results in its rapid uptake and internalization, but in the presence of heparin, rWnt5A just accumulates in the medium. B , PKC signaling is affected by high doses of heparin, regardless of whether rWnt5A is added. C , cells were treated with 2 milliunits/ml heparinase III for 24 h and examined for PKC signaling. PKC signaling is decreased upon heparinase III treatment and partially reconstituted upon the addition of rWnt5A. D , representative images of the motility assays using M93-047 cells demonstrating that heparinase III treatment decreased the rate of wound closure. E , graphical representation of UACC903 scratch closure rates demonstrating that rWnt5A addition can overcome heparinase III treatment. F , quantitative extracellular matrix invasion assays also demonstrate that heparinase III treatment decreases the rate of melanoma cell invasion, and this can be restored upon the addition of exogenous Wnt5A to the media of the cells ( n = 3; error bars show S.D.; **, p

    Article Snippet: Cells were serum-starved for 2 h and treated with 2 milliunits/ml heparinase III for 24 h and/or rWnt5A or rWnt3A for 16 h. Cells were then seeded at 5 × 105 cells/ml in the heparinase III/Wnt5A serum-free media in the transwell insert of a QCM plate (Millipore, Billerica, MA).

    Techniques: Binding Assay

    Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Journal: Molecular and Cellular Biology

    Article Title: The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

    doi:

    Figure Lengend Snippet: Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Article Snippet: Cell lysates from affinity-labeling samples were diluted at least 50-fold in a 0.1 M Tris acetate buffer and treated with heparitinase (code 100703; lot no. E95601; Seikagaku America, Rockville, Md.) and/or chondroitinase ABC (code 100332; lot no. KE95702; Seikagaku America) (final concentration, 10 mIU/ml) in 0.1 M Tris acetate buffer, pH 7.3, at 37°C for 1 h. In the case of treatment with heparitinase alone, shark cartilage chondroitin sulfate (catalog no. 2307; Calbiochem) was added to a final concentration of 50 μg/ml to protect the samples from digestion by chondroitinases possibly contaminating the heparitinase preparation.

    Techniques: Modification, Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation

    Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Journal: The Journal of Neuroscience

    Article Title: Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation

    doi: 10.1523/JNEUROSCI.19-04-01226.1999

    Figure Lengend Snippet: Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Article Snippet: Aliquots of the fractions were digested with nitrous acid or heparitinase (10 U/ml, 15 hr) to remove carbohydrate side chains or incubated with 50 μ m herbimycin A (Calbiochem, La Jolla, CA).

    Techniques: