Structured Review

Seikagaku heparitinase
Recombinant BAEBL/Fc binds to erythrocytes via HS and sialic acid. A , binding inhibition of BAEBL/Fc to enzyme-treated erythrocytes. Erythrocytes (10 5 cells) were pretreated with buffer; 0.0016, 0.016, 0.16, or 1.6 milliunits of <t>heparitinase;</t> or 0.004,
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Images

1) Product Images from "Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface *"

Article Title: Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface *

Journal:

doi: 10.1074/jbc.M109.021576

Recombinant BAEBL/Fc binds to erythrocytes via HS and sialic acid. A , binding inhibition of BAEBL/Fc to enzyme-treated erythrocytes. Erythrocytes (10 5 cells) were pretreated with buffer; 0.0016, 0.016, 0.16, or 1.6 milliunits of heparitinase; or 0.004,
Figure Legend Snippet: Recombinant BAEBL/Fc binds to erythrocytes via HS and sialic acid. A , binding inhibition of BAEBL/Fc to enzyme-treated erythrocytes. Erythrocytes (10 5 cells) were pretreated with buffer; 0.0016, 0.016, 0.16, or 1.6 milliunits of heparitinase; or 0.004,

Techniques Used: Recombinant, Binding Assay, Inhibition

HS-dependent binding is involved in merozoite invasion. A , inhibition of merozoite invasion of enzyme-treated erythrocytes. Erythrocytes (10 7 cells) were treated with buffer, with 0.016, 0.16, 1.6, or 16 milliunits ( mU ) of heparitinase, or with 0.04,
Figure Legend Snippet: HS-dependent binding is involved in merozoite invasion. A , inhibition of merozoite invasion of enzyme-treated erythrocytes. Erythrocytes (10 7 cells) were treated with buffer, with 0.016, 0.16, 1.6, or 16 milliunits ( mU ) of heparitinase, or with 0.04,

Techniques Used: Binding Assay, Inhibition

2) Product Images from "Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line"

Article Title: Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032351

Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line. DRMs were prepared from confluent PTr cells as described in Materials and Methods . Collected fractions, were concentrated, treated with heparitinase I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).
Figure Legend Snippet: Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line. DRMs were prepared from confluent PTr cells as described in Materials and Methods . Collected fractions, were concentrated, treated with heparitinase I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).

Techniques Used: Western Blot, Isolation, SDS Page, Staining, Concentration Assay

Identification of HSPGs expressed by a rat parathyroid cell line. A. RT-PCR analysis of PTr cells using syndecan-specific primers (see Materials and Methods for details). Total RNA was isolated from confluent cells and subjected to RT-PCR analysis. Amplified products were run on 2% agarose gel, stained with ethidium bromide and photographed under UV transilluminator. Lanes: M – 100 bp marker; SN1 – amplification with syndecan-1 specific primers; SN2 – amplification with syndecan-2 specific primers; SN3 – amplification with syndecan-3 specific primers; SN4 – amplification with syndecan-4 specific primers; G – amplification with GAPHD specific primers; (-) – negative controls containing no cDNA. B. Identification of HSPGs present in DRM fractions using WB analysis. Proteoglycans were isolated from confluent rat parathyroid cells and partially purified using Q-Sepharose anion-exchange chromatography. A proteoglycan-enriched fraction was incubated in the presence or absence of heparitinase I, subjected to SDS-PAGE and immunoblotted with anti-syndecan-1, anti-syndecan-4 or anti-ΔHS (3G10) antibodies. Lanes: 1, 4 and 7 represent the heparitinase I only; 2, 5 and 8 correspond to the control samples, incubated without heparitinase I; 3, 6, 8 correspond to the heparitinase-treated samples.
Figure Legend Snippet: Identification of HSPGs expressed by a rat parathyroid cell line. A. RT-PCR analysis of PTr cells using syndecan-specific primers (see Materials and Methods for details). Total RNA was isolated from confluent cells and subjected to RT-PCR analysis. Amplified products were run on 2% agarose gel, stained with ethidium bromide and photographed under UV transilluminator. Lanes: M – 100 bp marker; SN1 – amplification with syndecan-1 specific primers; SN2 – amplification with syndecan-2 specific primers; SN3 – amplification with syndecan-3 specific primers; SN4 – amplification with syndecan-4 specific primers; G – amplification with GAPHD specific primers; (-) – negative controls containing no cDNA. B. Identification of HSPGs present in DRM fractions using WB analysis. Proteoglycans were isolated from confluent rat parathyroid cells and partially purified using Q-Sepharose anion-exchange chromatography. A proteoglycan-enriched fraction was incubated in the presence or absence of heparitinase I, subjected to SDS-PAGE and immunoblotted with anti-syndecan-1, anti-syndecan-4 or anti-ΔHS (3G10) antibodies. Lanes: 1, 4 and 7 represent the heparitinase I only; 2, 5 and 8 correspond to the control samples, incubated without heparitinase I; 3, 6, 8 correspond to the heparitinase-treated samples.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Purification, Chromatography, Incubation, SDS Page

3) Product Images from "Effects of Modifiers of Glycosaminoglycan Biosynthesis on Outflow Facility in Perfusion Culture"

Article Title: Effects of Modifiers of Glycosaminoglycan Biosynthesis on Outflow Facility in Perfusion Culture

Journal: Investigative ophthalmology & visual science

doi: 10.1167/iovs.07-0903

Treatment of porcine anterior segments with GAGases. ( A ) Porcine anterior segments were treated in perfusion culture with chondroitinase ABC (0.25 U/mL), heparitinase (0.01 U/mL), or hyaluronidase (10 U/mL), individually or in combination. Control eyes
Figure Legend Snippet: Treatment of porcine anterior segments with GAGases. ( A ) Porcine anterior segments were treated in perfusion culture with chondroitinase ABC (0.25 U/mL), heparitinase (0.01 U/mL), or hyaluronidase (10 U/mL), individually or in combination. Control eyes

Techniques Used:

4) Product Images from "A homeostatic function of CXCR2 signalling in articular cartilage"

Article Title: A homeostatic function of CXCR2 signalling in articular cartilage

Journal: Annals of the Rheumatic Diseases

doi: 10.1136/annrheumdis-2014-205546

CXCL6 is present in healthy articular cartilage and its expression is associated with chondrocyte differentiation. (A) Immunofluorescence staining for CXCL6 (green) in normal and early osteoarthritis (moderate Mankin score) articular cartilage. Nuclei are stained using propidium iodide (red). Scale bar, 100 μm. (B) Densitometric quantification of CXCL6 staining (n=3). (C) Immunofluorescence staining for CXCL6 (red) in mouse articular cartilage of sham-operated control and destabilisation of the medial meniscus (DMM) operated mice, with 4′,6-diamidino-2-phenylindole staining the nuclei. Scale bar, 100 μm. (D) Densitometric quantification of CXCL6 staining (n=4). (E) Western blot analysis of CXCL6 release into supernatant from vehicle control or heparitinase treated, freeze-thawed wild-type mouse hip caps. (F) Real-time RT-PCR for CXCL6 mRNA in early and late passage human articular chondrocytes (n=3), *** p
Figure Legend Snippet: CXCL6 is present in healthy articular cartilage and its expression is associated with chondrocyte differentiation. (A) Immunofluorescence staining for CXCL6 (green) in normal and early osteoarthritis (moderate Mankin score) articular cartilage. Nuclei are stained using propidium iodide (red). Scale bar, 100 μm. (B) Densitometric quantification of CXCL6 staining (n=3). (C) Immunofluorescence staining for CXCL6 (red) in mouse articular cartilage of sham-operated control and destabilisation of the medial meniscus (DMM) operated mice, with 4′,6-diamidino-2-phenylindole staining the nuclei. Scale bar, 100 μm. (D) Densitometric quantification of CXCL6 staining (n=4). (E) Western blot analysis of CXCL6 release into supernatant from vehicle control or heparitinase treated, freeze-thawed wild-type mouse hip caps. (F) Real-time RT-PCR for CXCL6 mRNA in early and late passage human articular chondrocytes (n=3), *** p

Techniques Used: Expressing, Immunofluorescence, Staining, Mouse Assay, Western Blot, Quantitative RT-PCR

5) Product Images from "Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling"

Article Title: Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7601188

( A ) NRP1 is GAG modified on a single Ser 612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. ( B ) Multiple alignments of NRP1 from different species. Ser 612 is highly conserved among vertebrates. ( C ) NRP2, an NRP family member, is not GAG modified. ( D ) Design of siRNA and adenovirus constructs. Ser 612 exists in the bridge region between the b1b2 and MAM domains. ( E ) Replacement of Ser 612 by Ala 612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and chondroitinase (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125 I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e.
Figure Legend Snippet: ( A ) NRP1 is GAG modified on a single Ser 612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. ( B ) Multiple alignments of NRP1 from different species. Ser 612 is highly conserved among vertebrates. ( C ) NRP2, an NRP family member, is not GAG modified. ( D ) Design of siRNA and adenovirus constructs. Ser 612 exists in the bridge region between the b1b2 and MAM domains. ( E ) Replacement of Ser 612 by Ala 612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and chondroitinase (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125 I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e.

Techniques Used: Modification, Transfection, Mutagenesis, Construct, Binding Assay, Expressing, Plasmid Preparation, Incubation, Labeling, Immunoprecipitation, Radioactivity

A substantial fraction of cellular NRP1 is proteoglycan, composed of either HS or CS. ( A ) 125 I-labeled VEGF is crosslinked to different proteins in ECs and SMCs. Arrow indicates VEGF-binding protein specifically seen in SMCs. CASMC: coronary artery smooth muscle cell; BSMC: bronchial smooth muscle cell; HUVEC: human umbilical vein endothelial cell. ( B ) Western blots of exogenously expressed NRP1 in either CASMCs or HUVECs. Adenovirus encoding FLAG-tagged NRP1 was transfected 2 days before analysis at the indicated MOI. LacZ-encoding adenovirus was used as a control. ( C ) The high molecular weight band was not simply a covalently linked homodimer of NRP1. Only FLAG-tagged NRP1 or both FLAG-tagged and V5-tagged NRP1 were transfected in CASMCs, and the cell lysates were immunoprecipitated and detected by the indicated antibody. ( D ) Endogenous NRP1 was modified by GAG chain addition in both SMCs and ECs. The upper band in CASMC immunoprecipitates disappeared following treatment with both HSase and CSase. HUVEC-expressed NRP1 is also GAG modified. The band intensity was analyzed and the proportion of each glycanated form of NRP1 was determined. Data are from three separate experiments. HSase: heparitinase; CSase: chondroitinase.
Figure Legend Snippet: A substantial fraction of cellular NRP1 is proteoglycan, composed of either HS or CS. ( A ) 125 I-labeled VEGF is crosslinked to different proteins in ECs and SMCs. Arrow indicates VEGF-binding protein specifically seen in SMCs. CASMC: coronary artery smooth muscle cell; BSMC: bronchial smooth muscle cell; HUVEC: human umbilical vein endothelial cell. ( B ) Western blots of exogenously expressed NRP1 in either CASMCs or HUVECs. Adenovirus encoding FLAG-tagged NRP1 was transfected 2 days before analysis at the indicated MOI. LacZ-encoding adenovirus was used as a control. ( C ) The high molecular weight band was not simply a covalently linked homodimer of NRP1. Only FLAG-tagged NRP1 or both FLAG-tagged and V5-tagged NRP1 were transfected in CASMCs, and the cell lysates were immunoprecipitated and detected by the indicated antibody. ( D ) Endogenous NRP1 was modified by GAG chain addition in both SMCs and ECs. The upper band in CASMC immunoprecipitates disappeared following treatment with both HSase and CSase. HUVEC-expressed NRP1 is also GAG modified. The band intensity was analyzed and the proportion of each glycanated form of NRP1 was determined. Data are from three separate experiments. HSase: heparitinase; CSase: chondroitinase.

Techniques Used: Labeling, Binding Assay, Western Blot, Transfection, Molecular Weight, Immunoprecipitation, Modification

GAG modifications differentially affect NRP1 function in SMCs and ECs. ( A ) Experimental replacement of NRP1 in SMCs and ECs. After transfection with both N-G siRNA and adenoviral constructs, endogenous NRP1 was successfully replaced with either the glycanated form (NRP1 WT′) or non-glycanated form (NRP1 S612A) of NRP1. Tubulin was used as a loading control. ( B ) Addition of GAG to NRP1 enhances binding to VEGF in both types of cells. Two days after NRP1 replacement, cell lysates were immunoprecipitated with anti-FLAG antibody after incubation with 125 I-labeled VEGF (25 ng/ml) for 40 min at room temperature, and bound radioactivity was quantitated using a gamma counter. Heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding. Data are from three independent experiments. ( C ) VEGF (50 ng/ml) induced greater cell migration in SMCs expressing non-modified NRP1 S612A than those expressing NRP1 WT′. Migrated cells were quantified by counting cells in three random high-power fields (HPF, × 200). Similar results were obtained from additional two independent experiments. ( D ) VEGF (50 ng/ml) increased cell viability in ECs expressing NRP1 WT′ to a greater extent than in ECs expressing NRP1 S612A. Data are from three independent experiments. For panels B–D, error bars represent s.e. * P
Figure Legend Snippet: GAG modifications differentially affect NRP1 function in SMCs and ECs. ( A ) Experimental replacement of NRP1 in SMCs and ECs. After transfection with both N-G siRNA and adenoviral constructs, endogenous NRP1 was successfully replaced with either the glycanated form (NRP1 WT′) or non-glycanated form (NRP1 S612A) of NRP1. Tubulin was used as a loading control. ( B ) Addition of GAG to NRP1 enhances binding to VEGF in both types of cells. Two days after NRP1 replacement, cell lysates were immunoprecipitated with anti-FLAG antibody after incubation with 125 I-labeled VEGF (25 ng/ml) for 40 min at room temperature, and bound radioactivity was quantitated using a gamma counter. Heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding. Data are from three independent experiments. ( C ) VEGF (50 ng/ml) induced greater cell migration in SMCs expressing non-modified NRP1 S612A than those expressing NRP1 WT′. Migrated cells were quantified by counting cells in three random high-power fields (HPF, × 200). Similar results were obtained from additional two independent experiments. ( D ) VEGF (50 ng/ml) increased cell viability in ECs expressing NRP1 WT′ to a greater extent than in ECs expressing NRP1 S612A. Data are from three independent experiments. For panels B–D, error bars represent s.e. * P

Techniques Used: Transfection, Construct, Binding Assay, Immunoprecipitation, Incubation, Labeling, Radioactivity, Migration, Expressing, Modification

6) Product Images from "Distinct effects of glucose and glucosamine on vascular endothelial and smooth muscle cells: Evidence for a protective role for glucosamine in atherosclerosis"

Article Title: Distinct effects of glucose and glucosamine on vascular endothelial and smooth muscle cells: Evidence for a protective role for glucosamine in atherosclerosis

Journal: Cardiovascular Diabetology

doi: 10.1186/1475-2840-4-16

A. HSPG modulate LDL transport across EC monolayers. Endothelial cells were grown to confluence in tissue culture inserts (Falcon, 0.3 μm pore size) in 24 well plates to facilitate its access to the upper (luminal) and lower (subendothelial) surface of endothelial cells. The cells were incubated with medium alone (control) or medium containing 1 unit/ml each of heparinase and heparitinase in the bottom chamber for 2 h at 37°C. 125 I-LDL was then added to the cells in the upper chamber and the 125 I-LDL appeared in the media from the lower chamber was counted. Values represent Mean ± SD of triplicate measurements. B. Glucosamine treatment decreases LDL transport. Endothelial cells on tissue culture inserts were incubated with medium alone or medium containing 2.5 mM glucosamine for 16 h. 125 I-LDL transport was then determined as described above. Figure 3C. Monocyte adhesion to glucosamine treated endothelial cells decreases. Endothelial cells were grown to confluence in 24 well tissue culture plates. Cells were then incubated in medium or medium with glucosamine for 16 h. Subendothelial matrix was prepared from control and glucosamine treated endothelial cells and incubated with ( 3 H)leucine labeled THP-1 monocytes for 2 h. Unbound monocytes were washed four times with DMEM-BSA and the bound radioactivity was determined.
Figure Legend Snippet: A. HSPG modulate LDL transport across EC monolayers. Endothelial cells were grown to confluence in tissue culture inserts (Falcon, 0.3 μm pore size) in 24 well plates to facilitate its access to the upper (luminal) and lower (subendothelial) surface of endothelial cells. The cells were incubated with medium alone (control) or medium containing 1 unit/ml each of heparinase and heparitinase in the bottom chamber for 2 h at 37°C. 125 I-LDL was then added to the cells in the upper chamber and the 125 I-LDL appeared in the media from the lower chamber was counted. Values represent Mean ± SD of triplicate measurements. B. Glucosamine treatment decreases LDL transport. Endothelial cells on tissue culture inserts were incubated with medium alone or medium containing 2.5 mM glucosamine for 16 h. 125 I-LDL transport was then determined as described above. Figure 3C. Monocyte adhesion to glucosamine treated endothelial cells decreases. Endothelial cells were grown to confluence in 24 well tissue culture plates. Cells were then incubated in medium or medium with glucosamine for 16 h. Subendothelial matrix was prepared from control and glucosamine treated endothelial cells and incubated with ( 3 H)leucine labeled THP-1 monocytes for 2 h. Unbound monocytes were washed four times with DMEM-BSA and the bound radioactivity was determined.

Techniques Used: Incubation, Labeling, Radioactivity

7) Product Images from "Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells"

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

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI32412

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

Techniques Used: Expressing, Transfection, Incubation, Affinity Purification

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

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

8) Product Images from "Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements"

Article Title: Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200302152

Posttranslational modification of GPC3 in MDCK cells. Total proteoglycan or GPC3 isolated from stable transfectant cells was treated with heparitinase (Hase), chondroitinase ABC (Case), or endoglycosidase H (Endo H) as indicated, and fractionated by SDS-PAGE under reducing (DTT) or nonreducing conditions. (A) Two-subunit structure of GPC3. Western blots of total proteoglycan extract, using rat anti-HA mAb 3F10 to detect GPC3, and anti-ΔHS mAb 3G10 to detect the desaturated uronates that are generated by heparitinase and that remain in association with the core proteins. (B) Time course of the GPC3 maturation. Cells were pulse labeled with [ 35 S]cysteine-methionine for 10 min and chased for the indicated time periods. GPC3 from cell lysates, isolated using anti-HA antibody, was detected by autoradiography. Unreduced (left) and reduced (right) non enzyme-treated samples (top), and reduced glycosidase-treated samples (bottom) reveal that the HS substitution, proteolytic processing, and Endo H–resistant N-glycosylation of GPC3 follow similar time courses. (C) Inhibition of GPC3 processing by blocking ER export or calcium depletion. Cells were incubated for 6 h with (+) or without (−) 30 μM BFA or 2 μM A23187, pulse-labeled for 10 min, and chased for 60 min. GPC3 immunopurified from cell lysate was detected by autoradiography. (D) Endoproteolytic processing of GPC3ΔHS. Labeled HA-GPC3ΔHS was immunoprecipitated from the cell lysate (lanes 1 and 3) and the conditioned medium (lanes 2 and 4). Braces show glycanated GPC3, curved arrowheads indicate the GPC3 core protein, arrows indicates the ∼40-kD NH 2 -terminal (HA-tagged) α-subunit, and arrowheads indicate the COOH-terminal β subunit that is separated from the α-subunit by reduction. Numbers on the left represent molecular mass markers.
Figure Legend Snippet: Posttranslational modification of GPC3 in MDCK cells. Total proteoglycan or GPC3 isolated from stable transfectant cells was treated with heparitinase (Hase), chondroitinase ABC (Case), or endoglycosidase H (Endo H) as indicated, and fractionated by SDS-PAGE under reducing (DTT) or nonreducing conditions. (A) Two-subunit structure of GPC3. Western blots of total proteoglycan extract, using rat anti-HA mAb 3F10 to detect GPC3, and anti-ΔHS mAb 3G10 to detect the desaturated uronates that are generated by heparitinase and that remain in association with the core proteins. (B) Time course of the GPC3 maturation. Cells were pulse labeled with [ 35 S]cysteine-methionine for 10 min and chased for the indicated time periods. GPC3 from cell lysates, isolated using anti-HA antibody, was detected by autoradiography. Unreduced (left) and reduced (right) non enzyme-treated samples (top), and reduced glycosidase-treated samples (bottom) reveal that the HS substitution, proteolytic processing, and Endo H–resistant N-glycosylation of GPC3 follow similar time courses. (C) Inhibition of GPC3 processing by blocking ER export or calcium depletion. Cells were incubated for 6 h with (+) or without (−) 30 μM BFA or 2 μM A23187, pulse-labeled for 10 min, and chased for 60 min. GPC3 immunopurified from cell lysate was detected by autoradiography. (D) Endoproteolytic processing of GPC3ΔHS. Labeled HA-GPC3ΔHS was immunoprecipitated from the cell lysate (lanes 1 and 3) and the conditioned medium (lanes 2 and 4). Braces show glycanated GPC3, curved arrowheads indicate the GPC3 core protein, arrows indicates the ∼40-kD NH 2 -terminal (HA-tagged) α-subunit, and arrowheads indicate the COOH-terminal β subunit that is separated from the α-subunit by reduction. Numbers on the left represent molecular mass markers.

Techniques Used: Modification, Isolation, Transfection, SDS Page, Western Blot, Generated, Labeling, Autoradiography, Inhibition, Blocking Assay, Incubation, Immunoprecipitation

9) Product Images from "Preferential megalin-mediated transcytosis of low-hormonogenic thyroglobulin: A control mechanism for thyroid hormone release"

Article Title: Preferential megalin-mediated transcytosis of low-hormonogenic thyroglobulin: A control mechanism for thyroid hormone release

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

doi: 10.1073/pnas.2432267100

( a–c ) Effect of heparitinase, but not of chondroitinase ABC, on: ( a ) HSPGs expression (assessed by flow cytometry), ( b ) transcytosis of unlabeled Tgs, and ( c ) relation between HSPGs expression and transcytosis in FRTL-5 cells. ( d ) Effect of an antibody against the sequence 2489–2503 (anti-rTgP) on Tg transcytosis across FRTL-5 cells. Tgs were applied alone or with anti-rTgP or RIgG. ( e–h ) The sequence 2489–2503 is exposed to a greater extent in low-horm-rTg. Horm-rTg ( e and g ), and low-horm-rTg ( f and h ) were subjected to WB after preadsorption with RIgG (lanes 1 and bars 1), preadsorption with RIgG and precipitation with anti-rTgP (lanes 2 and bars 2), preadsorption with rabbit anti-T 4 (lanes 3 and bars 3), or preadsorption with anti-T 4 and precipitation with anti-rTgP (lanes 4 and bars 4). ( g and h ) Pixel density of the bands in e and f .
Figure Legend Snippet: ( a–c ) Effect of heparitinase, but not of chondroitinase ABC, on: ( a ) HSPGs expression (assessed by flow cytometry), ( b ) transcytosis of unlabeled Tgs, and ( c ) relation between HSPGs expression and transcytosis in FRTL-5 cells. ( d ) Effect of an antibody against the sequence 2489–2503 (anti-rTgP) on Tg transcytosis across FRTL-5 cells. Tgs were applied alone or with anti-rTgP or RIgG. ( e–h ) The sequence 2489–2503 is exposed to a greater extent in low-horm-rTg. Horm-rTg ( e and g ), and low-horm-rTg ( f and h ) were subjected to WB after preadsorption with RIgG (lanes 1 and bars 1), preadsorption with RIgG and precipitation with anti-rTgP (lanes 2 and bars 2), preadsorption with rabbit anti-T 4 (lanes 3 and bars 3), or preadsorption with anti-T 4 and precipitation with anti-rTgP (lanes 4 and bars 4). ( g and h ) Pixel density of the bands in e and f .

Techniques Used: Expressing, Flow Cytometry, Cytometry, Sequencing, Western Blot

10) Product Images from "Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines"

Article Title: Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

Journal: Virology Journal

doi: 10.1186/1743-422X-8-523

PvRII binding to heparitinase treated DARC+ erythrocytes . DARC+ human erythrocytes were digested with heparitinase I, an endoglycosidase specific for heparan sulfate. The number of rosettes of heparitinase-treated erythrocytes on COS-7 cells transfected with the pvRII expression vector, pHVDR22, is shown as the mean of three separate treatments.
Figure Legend Snippet: PvRII binding to heparitinase treated DARC+ erythrocytes . DARC+ human erythrocytes were digested with heparitinase I, an endoglycosidase specific for heparan sulfate. The number of rosettes of heparitinase-treated erythrocytes on COS-7 cells transfected with the pvRII expression vector, pHVDR22, is shown as the mean of three separate treatments.

Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation

11) Product Images from "Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity *"

Article Title: Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.177865

In situ activin A overlay assays on tissue sections. A–F , frozen sections of mouse E16.5 embryos were incubated with activin A NHis ( A–D ) or its mutant lacking the Lys 259 -Gly 277 segment ( E and F ), followed by detection of the bound proteins by immunohistochemistry. Sections treated with heparitinase ( C and D ) were included in the overlay assays to confirm that the signals were heparan sulfate-dependent. G and H , additional sections were subjected to immunohistochemistry with anti-heparan sulfate mAb 10E4 to clarify the localizations of heparan sulfate chains. The signals for bound activin A are localized to the basement membranes of various organs including the lungs ( A ) and skin ( B ), where the epidermis and hair follicles are labeled with arrowheads and asterisks , respectively. Scale bar , 100 μm.
Figure Legend Snippet: In situ activin A overlay assays on tissue sections. A–F , frozen sections of mouse E16.5 embryos were incubated with activin A NHis ( A–D ) or its mutant lacking the Lys 259 -Gly 277 segment ( E and F ), followed by detection of the bound proteins by immunohistochemistry. Sections treated with heparitinase ( C and D ) were included in the overlay assays to confirm that the signals were heparan sulfate-dependent. G and H , additional sections were subjected to immunohistochemistry with anti-heparan sulfate mAb 10E4 to clarify the localizations of heparan sulfate chains. The signals for bound activin A are localized to the basement membranes of various organs including the lungs ( A ) and skin ( B ), where the epidermis and hair follicles are labeled with arrowheads and asterisks , respectively. Scale bar , 100 μm.

Techniques Used: In Situ, Incubation, Mutagenesis, Immunohistochemistry, Labeling

12) Product Images from "The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains *"

Article Title: The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.430512

Affinity of proteoglycans from MC3T3-E1 cells to the hbd -CKFPTKRSKKAGRH 359 and their immunodetection. A , protein extracts from 5 × 10 6 MC3T3-E1 cells, undigested (−) or digested with heparitinase (+), were separated by SDS-PAGE (10% gel),
Figure Legend Snippet: Affinity of proteoglycans from MC3T3-E1 cells to the hbd -CKFPTKRSKKAGRH 359 and their immunodetection. A , protein extracts from 5 × 10 6 MC3T3-E1 cells, undigested (−) or digested with heparitinase (+), were separated by SDS-PAGE (10% gel),

Techniques Used: Immunodetection, SDS Page

Affinity of cell surface proteoglycans to chondroadherin and the hbd -CKFPTKRSKKAGRH 359 peptide from human chondrosarcoma 105kc cells. A , membrane proteins (from 5 × 10 6 105kc cells) were digested with heparitinase and separated by SDS-PAGE (14–12%
Figure Legend Snippet: Affinity of cell surface proteoglycans to chondroadherin and the hbd -CKFPTKRSKKAGRH 359 peptide from human chondrosarcoma 105kc cells. A , membrane proteins (from 5 × 10 6 105kc cells) were digested with heparitinase and separated by SDS-PAGE (14–12%

Techniques Used: SDS Page

13) Product Images from "The proteoglycan repertoire of lymphoid cells"

Article Title: The proteoglycan repertoire of lymphoid cells

Journal: Glycoconjugate Journal

doi: 10.1007/s10719-012-9427-9

Biosynthesis of [ 35 S]CS and HS in different subtypes of normal lymphocytes. Purified lymphocytes were cultured in vitro with and without PHA-L and labeled with 35 S-sulfate. 35 S-labeled macromolecules from medium (M) and cell (C) fractions were analyzed by gel chromatography after Chondroitinase-ABC and heparitinase or HNO2 treatment to determine the amount of CS ( white ) and HS ( black ). The incorporation into 35 S-labeled macromolecules is expressed per one million cells for all cell types used. The experiment was repeated three times and the results presented are from one representative experiment
Figure Legend Snippet: Biosynthesis of [ 35 S]CS and HS in different subtypes of normal lymphocytes. Purified lymphocytes were cultured in vitro with and without PHA-L and labeled with 35 S-sulfate. 35 S-labeled macromolecules from medium (M) and cell (C) fractions were analyzed by gel chromatography after Chondroitinase-ABC and heparitinase or HNO2 treatment to determine the amount of CS ( white ) and HS ( black ). The incorporation into 35 S-labeled macromolecules is expressed per one million cells for all cell types used. The experiment was repeated three times and the results presented are from one representative experiment

Techniques Used: Purification, Cell Culture, In Vitro, Labeling, Chromatography

14) Product Images from "Retroviral Gene Transfer Is Inhibited by Chondroitin Sulfate Proteoglycans/Glycosaminoglycans in Malignant Pleural Effusions *"

Article Title: Retroviral Gene Transfer Is Inhibited by Chondroitin Sulfate Proteoglycans/Glycosaminoglycans in Malignant Pleural Effusions *

Journal: The Journal of biological chemistry

doi:

Comparison of the inhibition of amphotropic retroviral transduction by untreated effusion supernatants, PG/GAGs isolated from effusions, or PG/GAGs isolated from the effusions that have been enzymatically treated with a variety of specific enzymes Shown is the relative inhibition to transduction conferred by supernatants from three malignant effusions, the PG/GAGs isolated with the D1 fraction (specific gravity 1.6 g/ml) from those effusions, or the PG/GAGs isolated with the D1 fraction that have been treated with a panel of specific degradative enzymes. A , 50% (v/v with LNPOZ vector stock) effusion supernatant; B , 50% D1 fraction; C , 50% D1 fraction plus Streptomyces hyaluronidase (10 units/ml); D , 50% D1 fraction plus bovine testicular hyaluronidase (250 units/ml); E , 50% D1 fraction plus chondroitinase AC (0.5 units/ml); F , D1 fraction plus chondroitinase ABC (0.5 units/ml); G , 50% D1 fraction plus heparinase (5 units/ml); H , 50% D1 fraction plus heparitinase (5 units/ml); I , 50% D1 fraction plus keratanase (1 unit/ml). n = 3 effusion specimens; data are presented as means ± S.E. *, p
Figure Legend Snippet: Comparison of the inhibition of amphotropic retroviral transduction by untreated effusion supernatants, PG/GAGs isolated from effusions, or PG/GAGs isolated from the effusions that have been enzymatically treated with a variety of specific enzymes Shown is the relative inhibition to transduction conferred by supernatants from three malignant effusions, the PG/GAGs isolated with the D1 fraction (specific gravity 1.6 g/ml) from those effusions, or the PG/GAGs isolated with the D1 fraction that have been treated with a panel of specific degradative enzymes. A , 50% (v/v with LNPOZ vector stock) effusion supernatant; B , 50% D1 fraction; C , 50% D1 fraction plus Streptomyces hyaluronidase (10 units/ml); D , 50% D1 fraction plus bovine testicular hyaluronidase (250 units/ml); E , 50% D1 fraction plus chondroitinase AC (0.5 units/ml); F , D1 fraction plus chondroitinase ABC (0.5 units/ml); G , 50% D1 fraction plus heparinase (5 units/ml); H , 50% D1 fraction plus heparitinase (5 units/ml); I , 50% D1 fraction plus keratanase (1 unit/ml). n = 3 effusion specimens; data are presented as means ± S.E. *, p

Techniques Used: Inhibition, Transduction, Isolation, Plasmid Preparation

15) Product Images from "Critical Role of Heparin Binding Domains of Ameloblastin for Dental Epithelium Cell Adhesion and Ameloblastoma Proliferation *"

Article Title: Critical Role of Heparin Binding Domains of Ameloblastin for Dental Epithelium Cell Adhesion and Ameloblastoma Proliferation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.033464

Deletion analysis of heparin binding regions of AMBN for heparin binding and cell adhesion. A , deletions of recombinant AMBN proteins. AB7 results from deletion of the first heparin binding region, and AB8 is the result of deletion of all three heparin binding regions. B , heparin binding of AB1, AB7, and AB8. AB7 slightly reduced heparin binding activity, but AB8 lost all activity. Heparin binding of AB1 was set at 100%. C , adhesion of HAT-7 cells to dishes coated with AB1, AB7, and AB8. AB7 had reduced cell binding activity. AB8 further reduced cell binding. Cells pretreated with heparitinase showed decreased binding to AB1. Cell adhesion of AB1 was set at 100% for comparison.
Figure Legend Snippet: Deletion analysis of heparin binding regions of AMBN for heparin binding and cell adhesion. A , deletions of recombinant AMBN proteins. AB7 results from deletion of the first heparin binding region, and AB8 is the result of deletion of all three heparin binding regions. B , heparin binding of AB1, AB7, and AB8. AB7 slightly reduced heparin binding activity, but AB8 lost all activity. Heparin binding of AB1 was set at 100%. C , adhesion of HAT-7 cells to dishes coated with AB1, AB7, and AB8. AB7 had reduced cell binding activity. AB8 further reduced cell binding. Cells pretreated with heparitinase showed decreased binding to AB1. Cell adhesion of AB1 was set at 100% for comparison.

Techniques Used: Binding Assay, Recombinant, Activity Assay, HAT Assay

16) Product Images from "Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Receptor Binding in Breast Carcinomas"

Article Title: Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Receptor Binding in Breast Carcinomas

Journal: The American Journal of Pathology

doi:

A–F: Reconstitution of FGF receptor complex in situ . A: Schematic representation of FGF-2 binding to tissue HSPGs in situ . HSPGs labeled “2” bind FGF-2, HSPGs labeled “1” fail to bind FGF-2. B: FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. Bound FGF-2 is detected with anti-FGF-2 mAb DE-6 and visualized with horseradish peroxidase. Note binding of FGF-2 to carcinoma cells (CA) and stroma, whereas a benign duct (ND) lacks FGF-2 binding. C: Negative control for FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. The tissue section was treated with heparitinase (20 mIU/ml, EC 4.2.2.8) before the growth factor incubation step. D: Schematic representation of FR1-AP binding to the FGF-2/HSPG complex in situ . Both HSPG classes labeled “2a” and “2b” bind FGF-2, but only HSPG 2b promotes the assembly of the complete receptor complex. E: FR1-AP (30 nmol/L) binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (10 nmol/L). Bound FR1-AP is detected with anti-AP antibody and visualized with horseradish peroxidase. Note FR1-AP binding to carcinoma cells and (syndecan-1-bearing) plasma cells (PC) and a normal duct lacking FR1-AP binding. F: Negative control for FR1-AP binding to infiltrating carcinoma. The FGF-2 incubation step was omitted. G–L: Immunohistochemical detection of syndecan core proteins: G: Syndecan-1 localization in a normal breast lobule with mAb BB4. Note high expression in normal epithelial cells (E) and in plasma cells. Inset shows normal breast acini (AC) labeled with anti-muscle-specific actin (MSA) antibody to highlight myoepithelial cells (ME). H: Syndecan-1 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas normal acini serve as internal positive control of the staining reaction. I : Syndecan-1 localization in a different infiltrating carcinoma. Note higher expression in the infiltrating carcinoma cells compared to a normal duct. J: Detection of syndecan-4 in a normal breast lobule with mAb 8G3. Note high levels of expression in normal acinar predominantly in a cytoplasmic location. Inset shows normal breast AC labeled with anti-MSA antibody to highlight MEs. K: Syndecan-4 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas a normal duct serves as internal positive control. L: Syndecan-4 localization in a different infiltrating carcinoma. Note high expression in the infiltrating carcinoma cells. M–O: Co-localization of FR1-AP binding and syndecan staining in adjacent sections of an infiltrating ductal carcinoma. M: FR1-AP binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (see description for E for details). N: Localization of syndecan-1. O: Localization of syndecan-4. Original magnifications, ×400.
Figure Legend Snippet: A–F: Reconstitution of FGF receptor complex in situ . A: Schematic representation of FGF-2 binding to tissue HSPGs in situ . HSPGs labeled “2” bind FGF-2, HSPGs labeled “1” fail to bind FGF-2. B: FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. Bound FGF-2 is detected with anti-FGF-2 mAb DE-6 and visualized with horseradish peroxidase. Note binding of FGF-2 to carcinoma cells (CA) and stroma, whereas a benign duct (ND) lacks FGF-2 binding. C: Negative control for FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. The tissue section was treated with heparitinase (20 mIU/ml, EC 4.2.2.8) before the growth factor incubation step. D: Schematic representation of FR1-AP binding to the FGF-2/HSPG complex in situ . Both HSPG classes labeled “2a” and “2b” bind FGF-2, but only HSPG 2b promotes the assembly of the complete receptor complex. E: FR1-AP (30 nmol/L) binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (10 nmol/L). Bound FR1-AP is detected with anti-AP antibody and visualized with horseradish peroxidase. Note FR1-AP binding to carcinoma cells and (syndecan-1-bearing) plasma cells (PC) and a normal duct lacking FR1-AP binding. F: Negative control for FR1-AP binding to infiltrating carcinoma. The FGF-2 incubation step was omitted. G–L: Immunohistochemical detection of syndecan core proteins: G: Syndecan-1 localization in a normal breast lobule with mAb BB4. Note high expression in normal epithelial cells (E) and in plasma cells. Inset shows normal breast acini (AC) labeled with anti-muscle-specific actin (MSA) antibody to highlight myoepithelial cells (ME). H: Syndecan-1 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas normal acini serve as internal positive control of the staining reaction. I : Syndecan-1 localization in a different infiltrating carcinoma. Note higher expression in the infiltrating carcinoma cells compared to a normal duct. J: Detection of syndecan-4 in a normal breast lobule with mAb 8G3. Note high levels of expression in normal acinar predominantly in a cytoplasmic location. Inset shows normal breast AC labeled with anti-MSA antibody to highlight MEs. K: Syndecan-4 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas a normal duct serves as internal positive control. L: Syndecan-4 localization in a different infiltrating carcinoma. Note high expression in the infiltrating carcinoma cells. M–O: Co-localization of FR1-AP binding and syndecan staining in adjacent sections of an infiltrating ductal carcinoma. M: FR1-AP binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (see description for E for details). N: Localization of syndecan-1. O: Localization of syndecan-4. Original magnifications, ×400.

Techniques Used: In Situ, Binding Assay, Labeling, Negative Control, Incubation, Immunohistochemistry, Expressing, Staining, Positive Control

Total amount of HSPGs in normal versus malignant epithelial cells: A: HSPGs regardless of core protein identity were localized on paraffin-embedded sections of breast carcinoma tissues by fluorescence microscopy with monoclonal antibody 3G10. The unsaturated uronate epitope recognized by this antibody (ΔHS) is generated within the HS chain by treatment with heparitinase (see Materials and Methods for details). B: Omission of this enzyme digestion step served as negative control. Alexa-546-conjugated secondary antibody was used for visualization. Original magnifications, ×400. Abbreviations: CA, carcinoma; ND, normal duct.
Figure Legend Snippet: Total amount of HSPGs in normal versus malignant epithelial cells: A: HSPGs regardless of core protein identity were localized on paraffin-embedded sections of breast carcinoma tissues by fluorescence microscopy with monoclonal antibody 3G10. The unsaturated uronate epitope recognized by this antibody (ΔHS) is generated within the HS chain by treatment with heparitinase (see Materials and Methods for details). B: Omission of this enzyme digestion step served as negative control. Alexa-546-conjugated secondary antibody was used for visualization. Original magnifications, ×400. Abbreviations: CA, carcinoma; ND, normal duct.

Techniques Used: Fluorescence, Microscopy, Generated, Negative Control

A: Characterization of HSPGs produced by the MCF-7 breast carcinoma cell line. HSPGs extracted from MCF-7 cells were treated with heparitinase and chondroitinase (see Materials and Methods) to degrade GAG chains and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 3.5 to 15% (w/v) gradient gel. Lane 1: cell equivalents stained with the anti-HS stub (ΔHS) antibody 3G10. Lane 2: cell equivalents stained with anti-syndecan-1 antibody BB4. Lane 3: cell equivalents stained with anti-syndecan-4 antibody 8G3 (see Materials and Methods for details). B: Fractionation of breast carcinoma cell HSPGs. HSPGs extracted from MCF-7 breast carcinoma cells were separated according to their ability to promote binding of FGF-2 to FGFR-1. The crude HSPG preparation was incubated with FGF-2 (final concentration, 16 nmol/L) and FR1-AP, immobilized on anti-AP-agarose beads. HSPGs present in the complex ( lane 2 ), in the supernatant ( lane 5 ) and HSPGs present before this fractionation ( lane 1 ) were analyzed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gel after the samples were digested with heparitinase and chondroitinase to remove all GAG chains (see Materials and Methods for details). The membrane was blotted with antibody 3G10 that detects all HSPGs regardless of the nature of the core protein. Controls include digestion of the HSPG preparation before complex formation ( lanes 3 and 6 ) and omission of FGF-2 from the binding reaction ( lanes 4 and 7 ). Bands identified by an asterisk cell equivalents in lane 1 cell equivalents in lanes 2 to 7 .
Figure Legend Snippet: A: Characterization of HSPGs produced by the MCF-7 breast carcinoma cell line. HSPGs extracted from MCF-7 cells were treated with heparitinase and chondroitinase (see Materials and Methods) to degrade GAG chains and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 3.5 to 15% (w/v) gradient gel. Lane 1: cell equivalents stained with the anti-HS stub (ΔHS) antibody 3G10. Lane 2: cell equivalents stained with anti-syndecan-1 antibody BB4. Lane 3: cell equivalents stained with anti-syndecan-4 antibody 8G3 (see Materials and Methods for details). B: Fractionation of breast carcinoma cell HSPGs. HSPGs extracted from MCF-7 breast carcinoma cells were separated according to their ability to promote binding of FGF-2 to FGFR-1. The crude HSPG preparation was incubated with FGF-2 (final concentration, 16 nmol/L) and FR1-AP, immobilized on anti-AP-agarose beads. HSPGs present in the complex ( lane 2 ), in the supernatant ( lane 5 ) and HSPGs present before this fractionation ( lane 1 ) were analyzed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gel after the samples were digested with heparitinase and chondroitinase to remove all GAG chains (see Materials and Methods for details). The membrane was blotted with antibody 3G10 that detects all HSPGs regardless of the nature of the core protein. Controls include digestion of the HSPG preparation before complex formation ( lanes 3 and 6 ) and omission of FGF-2 from the binding reaction ( lanes 4 and 7 ). Bands identified by an asterisk cell equivalents in lane 1 cell equivalents in lanes 2 to 7 .

Techniques Used: Produced, Polyacrylamide Gel Electrophoresis, Staining, Fractionation, Binding Assay, Incubation, Concentration Assay

17) Product Images from "Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?"

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

Journal: BMC Developmental Biology

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

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

Techniques Used: Negative Control, Incubation

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

Techniques Used: Staining, Immunohistochemistry

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

Techniques Used: In Situ, Staining, Immunohistochemistry, Incubation

18) Product Images from "Different Heparan Sulfate Proteoglycans Serve as Cellular Receptors for Human Papillomaviruses"

Article Title: Different Heparan Sulfate Proteoglycans Serve as Cellular Receptors for Human Papillomaviruses

Journal: Journal of Virology

doi: 10.1128/JVI.77.24.13125-13135.2003

Inhibition of HPV11 infection of human keratinocytes by treatment with heparitinase, but not by PLC. KH-SV cells were treated with heparitinase (1.9 U/ml) or PI-PLC (13 U/ml) or left untreated, as indicated. Subsequently, cells were incubated with HPV11 virions, incubated for 3 days, and analyzed for E1-E4 spliced viral RNA expression by RT-PCR. Amplicons were separated by gel electrophoresis and visualized by ethidium bromide staining. PCR in the absence of cDNA (H 2 O) and β-actin amplification served as specificity controls.
Figure Legend Snippet: Inhibition of HPV11 infection of human keratinocytes by treatment with heparitinase, but not by PLC. KH-SV cells were treated with heparitinase (1.9 U/ml) or PI-PLC (13 U/ml) or left untreated, as indicated. Subsequently, cells were incubated with HPV11 virions, incubated for 3 days, and analyzed for E1-E4 spliced viral RNA expression by RT-PCR. Amplicons were separated by gel electrophoresis and visualized by ethidium bromide staining. PCR in the absence of cDNA (H 2 O) and β-actin amplification served as specificity controls.

Techniques Used: Inhibition, Infection, Planar Chromatography, Incubation, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Staining, Polymerase Chain Reaction, Amplification

Inhibition of HPV16 VLP binding to human keratinocytes (KH-SV) by treatment with heparitinase, but not by PLC. Keratinocytes (KH-SV) were incubated with increasing concentrations of heparitinase or PI-PLC (13 U/ml) or left untreated, as indicated. Following a wash step with PBS, cells were incubated with (or without) VLP for 1 h and washed extensively, and cell-VLP complexes were analyzed by SDS-PAGE and Western blotting using MAb Camvir-1. The band at ∼45 kDa (indicated by a star) represents a cellular protein cross-reacting with MAb Camvir-1.
Figure Legend Snippet: Inhibition of HPV16 VLP binding to human keratinocytes (KH-SV) by treatment with heparitinase, but not by PLC. Keratinocytes (KH-SV) were incubated with increasing concentrations of heparitinase or PI-PLC (13 U/ml) or left untreated, as indicated. Following a wash step with PBS, cells were incubated with (or without) VLP for 1 h and washed extensively, and cell-VLP complexes were analyzed by SDS-PAGE and Western blotting using MAb Camvir-1. The band at ∼45 kDa (indicated by a star) represents a cellular protein cross-reacting with MAb Camvir-1.

Techniques Used: Inhibition, Binding Assay, Planar Chromatography, Incubation, SDS Page, Western Blot

Binding of HPV16 VLP to K562 cells and K562 transfectants expressing syndecan-1, syndecan-4, or glypican-1. Cell lines were incubated in the presence of HPV16 VLP or PBS for 1 h and washed extensively, and VLP in complex with cells were detected by immunoblotting using MAb Camvir-1. When indicated, cells were digested with heparitinase for 1 h to remove heparan sulfate from the cell surface prior to incubation with VLP. The double band at ∼50 to 55 kDa corresponds to posttranslational modification of L1. The band at ∼45 kDa (indicated by a star) represents a cellular protein cross-reacting with MAb Camvir-1 (in the absence of added VLP) that serves as an internal control for equal loading.
Figure Legend Snippet: Binding of HPV16 VLP to K562 cells and K562 transfectants expressing syndecan-1, syndecan-4, or glypican-1. Cell lines were incubated in the presence of HPV16 VLP or PBS for 1 h and washed extensively, and VLP in complex with cells were detected by immunoblotting using MAb Camvir-1. When indicated, cells were digested with heparitinase for 1 h to remove heparan sulfate from the cell surface prior to incubation with VLP. The double band at ∼50 to 55 kDa corresponds to posttranslational modification of L1. The band at ∼45 kDa (indicated by a star) represents a cellular protein cross-reacting with MAb Camvir-1 (in the absence of added VLP) that serves as an internal control for equal loading.

Techniques Used: Binding Assay, Expressing, Incubation, Modification

Transient infection of K562 cell lines expressing glypican-1 (K562 GP-1) or syndecan-1 (K562 SD-1) digested with PLC or heparitinase with native HPV11 virions (RT-PCR infectivity assay). Cells were treated with PI-PLC (13 U/ml) or heparitinase (1.9 U/ml) for 1 h or were left untreated, washed with PBS, infected with native HPV11 virions or mock infected, as indicated, and incubated for 3 days. Total RNA was isolated and E1-E4 transcripts were amplified by RT-PCR. Amplicons were separated by gel electrophoresis and visualized by ethidium bromide staining. Neutralization with an anti-HPV11 MAb (H11.B2) and amplification of β-actin served as controls, as indicated.
Figure Legend Snippet: Transient infection of K562 cell lines expressing glypican-1 (K562 GP-1) or syndecan-1 (K562 SD-1) digested with PLC or heparitinase with native HPV11 virions (RT-PCR infectivity assay). Cells were treated with PI-PLC (13 U/ml) or heparitinase (1.9 U/ml) for 1 h or were left untreated, washed with PBS, infected with native HPV11 virions or mock infected, as indicated, and incubated for 3 days. Total RNA was isolated and E1-E4 transcripts were amplified by RT-PCR. Amplicons were separated by gel electrophoresis and visualized by ethidium bromide staining. Neutralization with an anti-HPV11 MAb (H11.B2) and amplification of β-actin served as controls, as indicated.

Techniques Used: Infection, Expressing, Planar Chromatography, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Amplification, Nucleic Acid Electrophoresis, Staining, Neutralization

19) Product Images from "Serum amyloid A impairs the antiinflammatory properties of HDL"

Article Title: Serum amyloid A impairs the antiinflammatory properties of HDL

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI83475

HDL from AgNO 3 -injected mice colocalizes with the adipocyte cell surface. ( A ) HDL from PBS- and AgNO 3 -injected C57BL/6 mice was labeled with DiI (red). After exposure to these HDL preparations (50 μg protein/ml) for 6 hours, 3T3-L1 adipocytes were fixed in 2% formalin for 5 minutes. To distinguish the outside of cells from intracellular sites, the cell surface–associated ECM was stained with Alexa Fluor 488–conjugated WGA (green). Cell nuclei were counterstained with DAPI (blue). Cell morphology was shown by phase-contrast photography (left panels). Merged fluorescence (overlay) is shown in yellow. Original magnification, ×400. When the cell surface–associated ECM of 3T3-L1 adipocytes was digested with chondroitin ABC lyase and heparitinase for 1 hour prior to exposure to HDL, colocalization of HDL from AgNO 3 -injected mice with the cell surface (row 2, column 5) was lost and resulted in a similar distribution of dye to that seen with control HDL (row 3, column 5). ( B and C ) Enzyme digestion of the proteoglycans in the ECM also restored the impaired cholesterol efflux observed with HDL from AgNO 3 -injected mice ( B ) and Saa3 gene expression ( C ). Representative fluorescence images of 3 independent experiments are shown. Data represent mean ± SD. Data are representative of at least 3 independent experiments. * P
Figure Legend Snippet: HDL from AgNO 3 -injected mice colocalizes with the adipocyte cell surface. ( A ) HDL from PBS- and AgNO 3 -injected C57BL/6 mice was labeled with DiI (red). After exposure to these HDL preparations (50 μg protein/ml) for 6 hours, 3T3-L1 adipocytes were fixed in 2% formalin for 5 minutes. To distinguish the outside of cells from intracellular sites, the cell surface–associated ECM was stained with Alexa Fluor 488–conjugated WGA (green). Cell nuclei were counterstained with DAPI (blue). Cell morphology was shown by phase-contrast photography (left panels). Merged fluorescence (overlay) is shown in yellow. Original magnification, ×400. When the cell surface–associated ECM of 3T3-L1 adipocytes was digested with chondroitin ABC lyase and heparitinase for 1 hour prior to exposure to HDL, colocalization of HDL from AgNO 3 -injected mice with the cell surface (row 2, column 5) was lost and resulted in a similar distribution of dye to that seen with control HDL (row 3, column 5). ( B and C ) Enzyme digestion of the proteoglycans in the ECM also restored the impaired cholesterol efflux observed with HDL from AgNO 3 -injected mice ( B ) and Saa3 gene expression ( C ). Representative fluorescence images of 3 independent experiments are shown. Data represent mean ± SD. Data are representative of at least 3 independent experiments. * P

Techniques Used: Injection, Mouse Assay, Labeling, Staining, Whole Genome Amplification, Fluorescence, Expressing

20) Product Images from "Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Receptor Binding in Breast Carcinomas"

Article Title: Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Receptor Binding in Breast Carcinomas

Journal: The American Journal of Pathology

doi:

A–F: Reconstitution of FGF receptor complex in situ . A: Schematic representation of FGF-2 binding to tissue HSPGs in situ . HSPGs labeled “2” bind FGF-2, HSPGs labeled “1” fail to bind FGF-2. B: FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. Bound FGF-2 is detected with anti-FGF-2 mAb DE-6 and visualized with horseradish peroxidase. Note binding of FGF-2 to carcinoma cells (CA) and stroma, whereas a benign duct (ND) lacks FGF-2 binding. C: Negative control for FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. The tissue section was treated with heparitinase (20 mIU/ml, EC 4.2.2.8) before the growth factor incubation step. D: Schematic representation of FR1-AP binding to the FGF-2/HSPG complex in situ . Both HSPG classes labeled “2a” and “2b” bind FGF-2, but only HSPG 2b promotes the assembly of the complete receptor complex. E: FR1-AP (30 nmol/L) binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (10 nmol/L). Bound FR1-AP is detected with anti-AP antibody and visualized with horseradish peroxidase. Note FR1-AP binding to carcinoma cells and (syndecan-1-bearing) plasma cells (PC) and a normal duct lacking FR1-AP binding. F: Negative control for FR1-AP binding to infiltrating carcinoma. The FGF-2 incubation step was omitted. G–L: Immunohistochemical detection of syndecan core proteins: G: Syndecan-1 localization in a normal breast lobule with mAb BB4. Note high expression in normal epithelial cells (E) and in plasma cells. Inset shows normal breast acini (AC) labeled with anti-muscle-specific actin (MSA) antibody to highlight myoepithelial cells (ME). H: Syndecan-1 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas normal acini serve as internal positive control of the staining reaction. I : Syndecan-1 localization in a different infiltrating carcinoma. Note higher expression in the infiltrating carcinoma cells compared to a normal duct. J: Detection of syndecan-4 in a normal breast lobule with mAb 8G3. Note high levels of expression in normal acinar predominantly in a cytoplasmic location. Inset shows normal breast AC labeled with anti-MSA antibody to highlight MEs. K: Syndecan-4 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas a normal duct serves as internal positive control. L: Syndecan-4 localization in a different infiltrating carcinoma. Note high expression in the infiltrating carcinoma cells. M–O: Co-localization of FR1-AP binding and syndecan staining in adjacent sections of an infiltrating ductal carcinoma. M: FR1-AP binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (see description for E for details). N: Localization of syndecan-1. O: Localization of syndecan-4. Original magnifications, ×400.
Figure Legend Snippet: A–F: Reconstitution of FGF receptor complex in situ . A: Schematic representation of FGF-2 binding to tissue HSPGs in situ . HSPGs labeled “2” bind FGF-2, HSPGs labeled “1” fail to bind FGF-2. B: FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. Bound FGF-2 is detected with anti-FGF-2 mAb DE-6 and visualized with horseradish peroxidase. Note binding of FGF-2 to carcinoma cells (CA) and stroma, whereas a benign duct (ND) lacks FGF-2 binding. C: Negative control for FGF-2 (10 nmol/L) binding to HSPGs in section of infiltrating carcinoma. The tissue section was treated with heparitinase (20 mIU/ml, EC 4.2.2.8) before the growth factor incubation step. D: Schematic representation of FR1-AP binding to the FGF-2/HSPG complex in situ . Both HSPG classes labeled “2a” and “2b” bind FGF-2, but only HSPG 2b promotes the assembly of the complete receptor complex. E: FR1-AP (30 nmol/L) binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (10 nmol/L). Bound FR1-AP is detected with anti-AP antibody and visualized with horseradish peroxidase. Note FR1-AP binding to carcinoma cells and (syndecan-1-bearing) plasma cells (PC) and a normal duct lacking FR1-AP binding. F: Negative control for FR1-AP binding to infiltrating carcinoma. The FGF-2 incubation step was omitted. G–L: Immunohistochemical detection of syndecan core proteins: G: Syndecan-1 localization in a normal breast lobule with mAb BB4. Note high expression in normal epithelial cells (E) and in plasma cells. Inset shows normal breast acini (AC) labeled with anti-muscle-specific actin (MSA) antibody to highlight myoepithelial cells (ME). H: Syndecan-1 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas normal acini serve as internal positive control of the staining reaction. I : Syndecan-1 localization in a different infiltrating carcinoma. Note higher expression in the infiltrating carcinoma cells compared to a normal duct. J: Detection of syndecan-4 in a normal breast lobule with mAb 8G3. Note high levels of expression in normal acinar predominantly in a cytoplasmic location. Inset shows normal breast AC labeled with anti-MSA antibody to highlight MEs. K: Syndecan-4 localization in an infiltrating carcinoma. Note absence of staining in the infiltrating carcinoma cells, whereas a normal duct serves as internal positive control. L: Syndecan-4 localization in a different infiltrating carcinoma. Note high expression in the infiltrating carcinoma cells. M–O: Co-localization of FR1-AP binding and syndecan staining in adjacent sections of an infiltrating ductal carcinoma. M: FR1-AP binding to tissue section of infiltrating carcinoma previously incubated with FGF-2 (see description for E for details). N: Localization of syndecan-1. O: Localization of syndecan-4. Original magnifications, ×400.

Techniques Used: In Situ, Binding Assay, Labeling, Negative Control, Incubation, Immunohistochemistry, Expressing, Staining, Positive Control

Total amount of HSPGs in normal versus malignant epithelial cells: A: HSPGs regardless of core protein identity were localized on paraffin-embedded sections of breast carcinoma tissues by fluorescence microscopy with monoclonal antibody 3G10. The unsaturated uronate epitope recognized by this antibody (ΔHS) is generated within the HS chain by treatment with heparitinase (see Materials and Methods for details). B: Omission of this enzyme digestion step served as negative control. Alexa-546-conjugated secondary antibody was used for visualization. Original magnifications, ×400. Abbreviations: CA, carcinoma; ND, normal duct.
Figure Legend Snippet: Total amount of HSPGs in normal versus malignant epithelial cells: A: HSPGs regardless of core protein identity were localized on paraffin-embedded sections of breast carcinoma tissues by fluorescence microscopy with monoclonal antibody 3G10. The unsaturated uronate epitope recognized by this antibody (ΔHS) is generated within the HS chain by treatment with heparitinase (see Materials and Methods for details). B: Omission of this enzyme digestion step served as negative control. Alexa-546-conjugated secondary antibody was used for visualization. Original magnifications, ×400. Abbreviations: CA, carcinoma; ND, normal duct.

Techniques Used: Fluorescence, Microscopy, Generated, Negative Control

A: Characterization of HSPGs produced by the MCF-7 breast carcinoma cell line. HSPGs extracted from MCF-7 cells were treated with heparitinase and chondroitinase (see Materials and Methods) to degrade GAG chains and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 3.5 to 15% (w/v) gradient gel. Lane 1: cell equivalents stained with the anti-HS stub (ΔHS) antibody 3G10. Lane 2: cell equivalents stained with anti-syndecan-1 antibody BB4. Lane 3: cell equivalents stained with anti-syndecan-4 antibody 8G3 (see Materials and Methods for details). B: Fractionation of breast carcinoma cell HSPGs. HSPGs extracted from MCF-7 breast carcinoma cells were separated according to their ability to promote binding of FGF-2 to FGFR-1. The crude HSPG preparation was incubated with FGF-2 (final concentration, 16 nmol/L) and FR1-AP, immobilized on anti-AP-agarose beads. HSPGs present in the complex ( lane 2 ), in the supernatant ( lane 5 ) and HSPGs present before this fractionation ( lane 1 ) were analyzed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gel after the samples were digested with heparitinase and chondroitinase to remove all GAG chains (see Materials and Methods for details). The membrane was blotted with antibody 3G10 that detects all HSPGs regardless of the nature of the core protein. Controls include digestion of the HSPG preparation before complex formation ( lanes 3 and 6 ) and omission of FGF-2 from the binding reaction ( lanes 4 and 7 ). Bands identified by an asterisk cell equivalents in lane 1 cell equivalents in lanes 2 to 7 .
Figure Legend Snippet: A: Characterization of HSPGs produced by the MCF-7 breast carcinoma cell line. HSPGs extracted from MCF-7 cells were treated with heparitinase and chondroitinase (see Materials and Methods) to degrade GAG chains and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 3.5 to 15% (w/v) gradient gel. Lane 1: cell equivalents stained with the anti-HS stub (ΔHS) antibody 3G10. Lane 2: cell equivalents stained with anti-syndecan-1 antibody BB4. Lane 3: cell equivalents stained with anti-syndecan-4 antibody 8G3 (see Materials and Methods for details). B: Fractionation of breast carcinoma cell HSPGs. HSPGs extracted from MCF-7 breast carcinoma cells were separated according to their ability to promote binding of FGF-2 to FGFR-1. The crude HSPG preparation was incubated with FGF-2 (final concentration, 16 nmol/L) and FR1-AP, immobilized on anti-AP-agarose beads. HSPGs present in the complex ( lane 2 ), in the supernatant ( lane 5 ) and HSPGs present before this fractionation ( lane 1 ) were analyzed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gel after the samples were digested with heparitinase and chondroitinase to remove all GAG chains (see Materials and Methods for details). The membrane was blotted with antibody 3G10 that detects all HSPGs regardless of the nature of the core protein. Controls include digestion of the HSPG preparation before complex formation ( lanes 3 and 6 ) and omission of FGF-2 from the binding reaction ( lanes 4 and 7 ). Bands identified by an asterisk cell equivalents in lane 1 cell equivalents in lanes 2 to 7 .

Techniques Used: Produced, Polyacrylamide Gel Electrophoresis, Staining, Fractionation, Binding Assay, Incubation, Concentration Assay

21) Product Images from "Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor"

Article Title: Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-10-5

Confocal microscopy illustration of samples of MCF-7 cells A-C) without treatment; D-F) treated 30 minutes with heparitinase (in figure F a group of three cells), Cells were stained with ErbB4 carboxy-terminus specific HFR-1 antibody . Accumulation of immunoreactive perinuclear granules is indicated by arrows. G) Activity of Heparitinase was controlled by simultaneous Western analysis of ErbB4 cleavage from parallel samples.
Figure Legend Snippet: Confocal microscopy illustration of samples of MCF-7 cells A-C) without treatment; D-F) treated 30 minutes with heparitinase (in figure F a group of three cells), Cells were stained with ErbB4 carboxy-terminus specific HFR-1 antibody . Accumulation of immunoreactive perinuclear granules is indicated by arrows. G) Activity of Heparitinase was controlled by simultaneous Western analysis of ErbB4 cleavage from parallel samples.

Techniques Used: Confocal Microscopy, Staining, Activity Assay, Western Blot

A) The effects of heparitinase and PMA can be reverted by adding 40 μM TAPI-0 to the incubation medium . MCF-7 cells were treated for 30 minutes and Triton-X100 soluble lysates containing 40 μg protein were subjected to immunoblot. The position of full length ErbB4 protein and ErbB4 80 kDa fragment as detected by the polyclonal sc-283 anti ErbB4 antibody are indicated by arrows. B) The effect of heparitinase is specific for TACE-cleavable (JM-a) ErbB4 isoform. MCF-7 cells were transiently transfected with ErbB4 JM-a CYT2HA or JM-b CYT2HA gene construct. Lysates were subjected to immunoblot with HA-specific monoclonal antibody. C) The effect of heparitinase treatment could be demonstrated in T47D cells treated similarly to MCF-7 cells. D) Degradation of chondroitin sulfate did not increase the formation of ErbB4 80 kDa fragment in T47D cells. E) Heat inactivated incubation medium from heparitinase treatment of T47D cells had only small effect on ErbB4 80 kDa fragment formation. The intensity of the ErbB4 80 kDa fragment staining as indicated in C and D was quantified with ImageJ software vs. 1.38 (NIH, USA). Beta-actin was used as load control (not shown). Abbreviations: Htase, heparitinase; Ctase, chondroitinase, PMA, phorbol myristyl acetate. Ht med. heat inactivated heparitinase incubation medium. The images are representative of at least three independent analyses.
Figure Legend Snippet: A) The effects of heparitinase and PMA can be reverted by adding 40 μM TAPI-0 to the incubation medium . MCF-7 cells were treated for 30 minutes and Triton-X100 soluble lysates containing 40 μg protein were subjected to immunoblot. The position of full length ErbB4 protein and ErbB4 80 kDa fragment as detected by the polyclonal sc-283 anti ErbB4 antibody are indicated by arrows. B) The effect of heparitinase is specific for TACE-cleavable (JM-a) ErbB4 isoform. MCF-7 cells were transiently transfected with ErbB4 JM-a CYT2HA or JM-b CYT2HA gene construct. Lysates were subjected to immunoblot with HA-specific monoclonal antibody. C) The effect of heparitinase treatment could be demonstrated in T47D cells treated similarly to MCF-7 cells. D) Degradation of chondroitin sulfate did not increase the formation of ErbB4 80 kDa fragment in T47D cells. E) Heat inactivated incubation medium from heparitinase treatment of T47D cells had only small effect on ErbB4 80 kDa fragment formation. The intensity of the ErbB4 80 kDa fragment staining as indicated in C and D was quantified with ImageJ software vs. 1.38 (NIH, USA). Beta-actin was used as load control (not shown). Abbreviations: Htase, heparitinase; Ctase, chondroitinase, PMA, phorbol myristyl acetate. Ht med. heat inactivated heparitinase incubation medium. The images are representative of at least three independent analyses.

Techniques Used: Incubation, Transfection, Construct, Staining, Software

Exogeneous heparin or HS inhibit heparitinase-induced ErbB4 80 kDa fragment formation . A) Small concentrations of heparin slightly enhance the heparitinase-induced ErbB4 80 kDa fragment formation, whereas 1 μg/ml heparin and higher concentrations inhibit ErbB4 80 kDa fragment formation as indicated in lysates of T47D cells. B) Incubation with increasing concentrations of bovine lung heparin only did not have marked effect on cells. N. s., non-specific staining in A and B. C) Both heparin and heparan sulfate inhibited activity of recombinant TACE at high concentrations but displayed some enhancement of enzyme activity at low concentrations D) Heparitinase treatment of living MCF-7 cells enhanced cleavage of fluorescent TACE substrate peptide (p = 6 × 10 -11 ) and the effect of heparitinase could be largely reverted by adding 40 μM TAPI-0 to the incubation medium (p = 10 -5 ). The p-values were calculated with two-tailed pairwise Student's test comparing all time points. The enzyme activity analysis was performed three times with similar results. The data shown represents results from a single assay.
Figure Legend Snippet: Exogeneous heparin or HS inhibit heparitinase-induced ErbB4 80 kDa fragment formation . A) Small concentrations of heparin slightly enhance the heparitinase-induced ErbB4 80 kDa fragment formation, whereas 1 μg/ml heparin and higher concentrations inhibit ErbB4 80 kDa fragment formation as indicated in lysates of T47D cells. B) Incubation with increasing concentrations of bovine lung heparin only did not have marked effect on cells. N. s., non-specific staining in A and B. C) Both heparin and heparan sulfate inhibited activity of recombinant TACE at high concentrations but displayed some enhancement of enzyme activity at low concentrations D) Heparitinase treatment of living MCF-7 cells enhanced cleavage of fluorescent TACE substrate peptide (p = 6 × 10 -11 ) and the effect of heparitinase could be largely reverted by adding 40 μM TAPI-0 to the incubation medium (p = 10 -5 ). The p-values were calculated with two-tailed pairwise Student's test comparing all time points. The enzyme activity analysis was performed three times with similar results. The data shown represents results from a single assay.

Techniques Used: Incubation, Staining, Activity Assay, Recombinant, Two Tailed Test

A) TACE antibody was shown immunoprecipitate Syndecan-4 shown as high molecular weight smear in immunoblot by anti Syndecan-4 . The co-immunoprecipitation was abolished by heparitinase treatment. B) Syndecan-4 and TACE colocalize in MCF-7 cells. The colocalization is disrupted by heparitinase treatment.
Figure Legend Snippet: A) TACE antibody was shown immunoprecipitate Syndecan-4 shown as high molecular weight smear in immunoblot by anti Syndecan-4 . The co-immunoprecipitation was abolished by heparitinase treatment. B) Syndecan-4 and TACE colocalize in MCF-7 cells. The colocalization is disrupted by heparitinase treatment.

Techniques Used: Molecular Weight, Immunoprecipitation

22) Product Images from "Defects in the Medial Entorhinal Cortex and Dentate Gyrus in the Mouse Model of Sanfilippo Syndrome Type B"

Article Title: Defects in the Medial Entorhinal Cortex and Dentate Gyrus in the Mouse Model of Sanfilippo Syndrome Type B

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027461

Elevated level of heparan sulfate proteoglycan in the MEC region of the MPS IIIB brain. Sections were stained, as indicated, with antibodies against epitopes in the protein core of glypican 1 and glypican 5 and against the carbohydrate neo-epitope 3G10 formed by pre-treatment of the proteoglycan with heparitinase. The top row shows prominent staining in the MEC region of 6 months old Naglu −/− mice, the middle row shows absence of such staining in the LEC region of these mice, and the bottom row, absence of staining in the MEC region of age-matched Naglu +/− (control) mice. The larger image was taken at 10X magnification, and the insert at 40X. The scale bar, 200 µm, applies to all figures in the panel.
Figure Legend Snippet: Elevated level of heparan sulfate proteoglycan in the MEC region of the MPS IIIB brain. Sections were stained, as indicated, with antibodies against epitopes in the protein core of glypican 1 and glypican 5 and against the carbohydrate neo-epitope 3G10 formed by pre-treatment of the proteoglycan with heparitinase. The top row shows prominent staining in the MEC region of 6 months old Naglu −/− mice, the middle row shows absence of such staining in the LEC region of these mice, and the bottom row, absence of staining in the MEC region of age-matched Naglu +/− (control) mice. The larger image was taken at 10X magnification, and the insert at 40X. The scale bar, 200 µm, applies to all figures in the panel.

Techniques Used: Staining, Mouse Assay

23) Product Images from "Cysteine-Rich Domain of Human ADAM 12 (Meltrin ?) Supports Tumor Cell Adhesion"

Article Title: Cysteine-Rich Domain of Human ADAM 12 (Meltrin ?) Supports Tumor Cell Adhesion

Journal: The American Journal of Pathology

doi:

MDA-MB-231 cell attachment to rADAM 12-cys is mediated through a cell surface heparan sulfate proteoglycan. A shows the effect of function-blocking β1 integrin antibodies on attachment to rADAM 12-cys and to EHS-laminin of MDA-MB-231 cells. MDA-MB-231 cells on rADAM 12-cys (-•-) and on EHS-laminin (-○-). Note that the antibodies completely inhibited attachment to laminin as expected, but that no dose response was obtained on attaching to rADAM 12-cys. B shows that MDA-MB-231 cells grown in the presence of 20 mmol/L sodium chlorate ( hatched bars ) are inhibited in their cell attachment on rADAM 12-cys compared with cultures without the addition of sodium chlorate ( open bars ) or with the addition of 10 mmol/L sodium sulfate ( cross-hatched bars ). No effect of sodium chlorate ( hatched bars ) on cell attachment to laminin compared with untreated cells ( open bars ) was observed. Data present the mean ± SEM. C demonstrates the effect of 10 μg/ml each of heparin, heparan, chondroitin sulfate A, B, C, and hyaluronic acid as well as 1 mU/ml heparitinase or 50 mU/ml chondroitinase ABC. Note that MDA-MB-231 cell attachment is sensitive to heparin, heparan, and heparitinase. D demonstrates a dose response of heparin on cell attachment to rADAM 12-cys (-•-) and to laminin (-○-). Note that low doses of heparin inhibited the attachment of MDA-MB-231 cells. No effect of heparin was observed on cell attachment to laminin. Data present the mean ± SEM.
Figure Legend Snippet: MDA-MB-231 cell attachment to rADAM 12-cys is mediated through a cell surface heparan sulfate proteoglycan. A shows the effect of function-blocking β1 integrin antibodies on attachment to rADAM 12-cys and to EHS-laminin of MDA-MB-231 cells. MDA-MB-231 cells on rADAM 12-cys (-•-) and on EHS-laminin (-○-). Note that the antibodies completely inhibited attachment to laminin as expected, but that no dose response was obtained on attaching to rADAM 12-cys. B shows that MDA-MB-231 cells grown in the presence of 20 mmol/L sodium chlorate ( hatched bars ) are inhibited in their cell attachment on rADAM 12-cys compared with cultures without the addition of sodium chlorate ( open bars ) or with the addition of 10 mmol/L sodium sulfate ( cross-hatched bars ). No effect of sodium chlorate ( hatched bars ) on cell attachment to laminin compared with untreated cells ( open bars ) was observed. Data present the mean ± SEM. C demonstrates the effect of 10 μg/ml each of heparin, heparan, chondroitin sulfate A, B, C, and hyaluronic acid as well as 1 mU/ml heparitinase or 50 mU/ml chondroitinase ABC. Note that MDA-MB-231 cell attachment is sensitive to heparin, heparan, and heparitinase. D demonstrates a dose response of heparin on cell attachment to rADAM 12-cys (-•-) and to laminin (-○-). Note that low doses of heparin inhibited the attachment of MDA-MB-231 cells. No effect of heparin was observed on cell attachment to laminin. Data present the mean ± SEM.

Techniques Used: Multiple Displacement Amplification, Cell Attachment Assay, Blocking Assay

24) Product Images from "Glypican-1 and α4(V) Collagen Are Required for Schwann Cell Myelination"

Article Title: Glypican-1 and α4(V) Collagen Are Required for Schwann Cell Myelination

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2544-05.2006

siRNA-mediated suppression of target gene expression in Schwann cells. A , Detergent extracts of siNeg- or siGly1-transfected Schwann cells were digested with heparitinase and subjected to immunoblot analysis with anti-glypican-1 antibodies (top); aliquots of undigested extracts were immunoblotted with anti-actin antibodies (bottom) to verify equal sample loading. B , Cell surface expression of glypican-1 was assessed by immunofluorescent staining of transfected cells 48 h after transfection using anti-glypican-1 antibodies (green) and DAPI (4′,6′-diamidino-2-phenylindole) to stain nuclei (red). C , Schwann cells were transfected with siNeg or siα4(V), and conditioned media and cell lysates were harvested 48 h later (left) or at the indicated times after transfection (right); aliquots of medium (top) or cell lysates (bottom) were subjected to immunoblot analysis and stained with anti-α4(V) collagen (top) or anti-β-actin (bottom) antibodies.
Figure Legend Snippet: siRNA-mediated suppression of target gene expression in Schwann cells. A , Detergent extracts of siNeg- or siGly1-transfected Schwann cells were digested with heparitinase and subjected to immunoblot analysis with anti-glypican-1 antibodies (top); aliquots of undigested extracts were immunoblotted with anti-actin antibodies (bottom) to verify equal sample loading. B , Cell surface expression of glypican-1 was assessed by immunofluorescent staining of transfected cells 48 h after transfection using anti-glypican-1 antibodies (green) and DAPI (4′,6′-diamidino-2-phenylindole) to stain nuclei (red). C , Schwann cells were transfected with siNeg or siα4(V), and conditioned media and cell lysates were harvested 48 h later (left) or at the indicated times after transfection (right); aliquots of medium (top) or cell lysates (bottom) were subjected to immunoblot analysis and stained with anti-α4(V) collagen (top) or anti-β-actin (bottom) antibodies.

Techniques Used: Expressing, Transfection, Staining

25) Product Images from "The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification"

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

Journal: Molecular and Cellular Biology

doi:

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.
Figure Legend 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.

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

26) Product Images from "Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins"

Article Title: Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-2-18

Inhibition of III1-C mediated ERK activation by heparitinase. RASMCs were collected in DMEM+0.5% BSA and were then treated in suspension with either no GAGase ((-), PDGF, FN, C, Hep, and 11C samples) or with 0.1 u/ml of heparitinase (H'ase), or 0.1 u/ml of chondroitinase ABC (C'ase) for 1 hr at 37°C. Cells were then either left in suspension in the absence (-) or presence of PDGF (PDGF) for 10 min, or cells were plated onto dishes precoated with either fibronectin (FN), III1-C (C, Hep, H'ase, and C'ase lanes), or III 11-C (11C). One sample of cells plated onto III1-C contained 100 μg/ml heparin (Hep) in the medium. Cell samples were analyzed by immunoblotting with anti-phospho-p44/42 MAPK antibodies (Phos-ERK panel) followed by anti-ERK antibodies (ERK panel) as described in the legend to Fig. 5 . The experiment was performed four times with similar results each time.
Figure Legend Snippet: Inhibition of III1-C mediated ERK activation by heparitinase. RASMCs were collected in DMEM+0.5% BSA and were then treated in suspension with either no GAGase ((-), PDGF, FN, C, Hep, and 11C samples) or with 0.1 u/ml of heparitinase (H'ase), or 0.1 u/ml of chondroitinase ABC (C'ase) for 1 hr at 37°C. Cells were then either left in suspension in the absence (-) or presence of PDGF (PDGF) for 10 min, or cells were plated onto dishes precoated with either fibronectin (FN), III1-C (C, Hep, H'ase, and C'ase lanes), or III 11-C (11C). One sample of cells plated onto III1-C contained 100 μg/ml heparin (Hep) in the medium. Cell samples were analyzed by immunoblotting with anti-phospho-p44/42 MAPK antibodies (Phos-ERK panel) followed by anti-ERK antibodies (ERK panel) as described in the legend to Fig. 5 . The experiment was performed four times with similar results each time.

Techniques Used: Inhibition, Activation Assay

Affinity chromatography of 35 SO 4 -labeled proteoglycans on III1-C Sepharose. Panel A. RASMCs were labeled with 35 SO 4 , which preferentially labels the glycosaminoglycan chains of proteoglycans. Cells were lysed in NP40 buffer and lysates were applied to either III1-C Sepharose (C lanes) or III 11-C Sepharose (11C lanes) columns. The flow through fractions were collected (Flow Thru lanes), the columns were washed, and the bound material was eluted by first applying 8 M urea elution buffer (Urea lanes), washing the columns with PBS, then collecting the Sepharose beads and boiling them in SDS-PAGE sample buffer (SDS lanes). The lane labeled St shows a sample of the starting material. All samples were separated on SDS-PAGE gels then detected by phosphorimager analysis. Numbers to the left of the panel indicate the migration of molecular mass markers (kDa). Panel B. Samples of the starting material were treated with either no enzyme ((-)), or with 0.03 u/ml chondroitinase ABC (C'ase), or 0.03 u/ml heparitinase (H'ase), then analyzed by SDS-PAGE and phosphorimager. The experiment was performed twice with similar results both times.
Figure Legend Snippet: Affinity chromatography of 35 SO 4 -labeled proteoglycans on III1-C Sepharose. Panel A. RASMCs were labeled with 35 SO 4 , which preferentially labels the glycosaminoglycan chains of proteoglycans. Cells were lysed in NP40 buffer and lysates were applied to either III1-C Sepharose (C lanes) or III 11-C Sepharose (11C lanes) columns. The flow through fractions were collected (Flow Thru lanes), the columns were washed, and the bound material was eluted by first applying 8 M urea elution buffer (Urea lanes), washing the columns with PBS, then collecting the Sepharose beads and boiling them in SDS-PAGE sample buffer (SDS lanes). The lane labeled St shows a sample of the starting material. All samples were separated on SDS-PAGE gels then detected by phosphorimager analysis. Numbers to the left of the panel indicate the migration of molecular mass markers (kDa). Panel B. Samples of the starting material were treated with either no enzyme ((-)), or with 0.03 u/ml chondroitinase ABC (C'ase), or 0.03 u/ml heparitinase (H'ase), then analyzed by SDS-PAGE and phosphorimager. The experiment was performed twice with similar results both times.

Techniques Used: Affinity Chromatography, Labeling, Flow Cytometry, SDS Page, Migration

Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.
Figure Legend Snippet: Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.

Techniques Used: Affinity Chromatography, Flow Cytometry, SDS Page

27) Product Images from "Mammalian and Drosophila cells adhere to the laminin ?4 LG4 domain through syndecans, but not glypicans"

Article Title: Mammalian and Drosophila cells adhere to the laminin ?4 LG4 domain through syndecans, but not glypicans

Journal: Biochemical Journal

doi: 10.1042/BJ20040558

Inhibition of cell adhesion to GST–LG4 by heparin, heparan sulphate and heparitinase I
Figure Legend Snippet: Inhibition of cell adhesion to GST–LG4 by heparin, heparan sulphate and heparitinase I

Techniques Used: Inhibition

28) Product Images from "Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2"

Article Title: Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2

Journal: Journal of Cell Communication and Signaling

doi: 10.1007/s12079-008-0018-2

Decorin and biglycan, but not HSPGs synthesis is increased in mdx muscle fibroblasts. a Samples obtained from conditioned medium, corresponding to equal amounts of total cell extract proteins, of mdx and control fibroblasts were concentrated and subjected to SDS-PAGE followed by fluorography. On the left , upper and lower brackets indicate CSPG (biglycan and decorin, respectively). Sample treatments included: heparitinase ( H ase ) and chondroitinase ABC ( C ABC ). b Samples similar to a were concentrated and incubated with C ABC , separated by SDS gel electrophoresis, transferred to nitrocellulose membranes, stained with specific antibodies against decorin to reveal decorin core protein using enhanced chemiluminescence. c Samples obtained from cell extract, corresponding to equal amounts of total proteins were concentrated and subjected to SDS-PAGE followed by fluorography, the bracket indicates HSPGs. d Samples similar to c . were incubated with Hase, separated by SDS-PAGE, transferred to nitrocellulose membranes and stained with anti-stub specific monoclonal antibodies (3G10) to reveal the core proteins of HSPGs by enhanced chemiluminescence. Based on the molecular weights of the core proteins, it is possible to identify the following PGs: syndecan-3 (Syn-3); syndecan-1 (Syn-1); glypican (Gly); syndecan-2 (Syn-2) and syndecan-4 (Syn-4)
Figure Legend Snippet: Decorin and biglycan, but not HSPGs synthesis is increased in mdx muscle fibroblasts. a Samples obtained from conditioned medium, corresponding to equal amounts of total cell extract proteins, of mdx and control fibroblasts were concentrated and subjected to SDS-PAGE followed by fluorography. On the left , upper and lower brackets indicate CSPG (biglycan and decorin, respectively). Sample treatments included: heparitinase ( H ase ) and chondroitinase ABC ( C ABC ). b Samples similar to a were concentrated and incubated with C ABC , separated by SDS gel electrophoresis, transferred to nitrocellulose membranes, stained with specific antibodies against decorin to reveal decorin core protein using enhanced chemiluminescence. c Samples obtained from cell extract, corresponding to equal amounts of total proteins were concentrated and subjected to SDS-PAGE followed by fluorography, the bracket indicates HSPGs. d Samples similar to c . were incubated with Hase, separated by SDS-PAGE, transferred to nitrocellulose membranes and stained with anti-stub specific monoclonal antibodies (3G10) to reveal the core proteins of HSPGs by enhanced chemiluminescence. Based on the molecular weights of the core proteins, it is possible to identify the following PGs: syndecan-3 (Syn-3); syndecan-1 (Syn-1); glypican (Gly); syndecan-2 (Syn-2) and syndecan-4 (Syn-4)

Techniques Used: SDS Page, Incubation, SDS-Gel, Electrophoresis, Staining

29) Product Images from "Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *"

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.686295

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

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

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

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

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

Techniques Used:

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

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

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

Techniques Used: Confocal Microscopy, Flow Cytometry, Cytometry

30) Product Images from "Recruitment of Mammalian Cell Fibronectin to the Surface of Chlamydia trachomatis"

Article Title: Recruitment of Mammalian Cell Fibronectin to the Surface of Chlamydia trachomatis

Journal: Infection and Immunity

doi: 10.1128/IAI.70.7.3935-3938.2002

Sensitivity of EB-associated fibronectin to heparitinase. C. trachomatis serovar D EB were incubated in the presence of heparitinase or chondroitinase for 30 min and washed with PBS. Fibronectin in the EB-containing pellet and the supernatant was detected by immunoblotting. The contents of all lanes were incubated with rabbit anti-fibronectin antibody and detected with goat anti-rabbit HRP conjugated antibody.
Figure Legend Snippet: Sensitivity of EB-associated fibronectin to heparitinase. C. trachomatis serovar D EB were incubated in the presence of heparitinase or chondroitinase for 30 min and washed with PBS. Fibronectin in the EB-containing pellet and the supernatant was detected by immunoblotting. The contents of all lanes were incubated with rabbit anti-fibronectin antibody and detected with goat anti-rabbit HRP conjugated antibody.

Techniques Used: Incubation

31) Product Images from "Roles of two types of heparan sulfate clusters in Wnt distribution and signaling in Xenopus"

Article Title: Roles of two types of heparan sulfate clusters in Wnt distribution and signaling in Xenopus

Journal: Nature Communications

doi: 10.1038/s41467-017-02076-0

Endogenous Wnt8 shows not only a gradient but also HS-dependent punctate distributions. a The gradient of the endogenous Wnt8 protein from the lateral to mid-dorsal marginal zones at the mid-gastrula stage (st. 11.5). The observed region is indicated by the cyan box (reported localization of wnt8 mRNA in magenta). Embryos were flat-mounted under a coverslip and the image was acquired using automatic tiling with the maximum intensity projection of z -stacks (junctions of tiling appeared darker, arrowheads). b Quantification of the gradient of Wnt8 staining ( a ). The junctions of tiling are indicated by dotted lines, showing the drop in intensity. c A high-resolution image of endogenous Wnt8 staining in the VMZ. wnt8 is expressed in the underlying mesoderm, but not in the superficial layer in Xenopus embryos. An optical section at the subapical region of cells (basal to the tight junction) of the superficial layer is shown (see also Supplementary Fig. 2b, d ). d Exogenous mV-Wnt8 expression and heparitinase treatment. Experimental procedures are illustrated on the left. Embryos were observed at stage 10.5. The source cells are indicated with “*”. e Immunostaining of HS chains with NAH46 (for N- acetyl HS) or HepSS-1 (for N- sulfo HS). Notably HepSS-1 staining shows puncta inside cells (orange arrowheads). f – k The colocalization of endogenous Wnt8 and a subpopulation of HS. Gastrula embryos (st. 10.5) were co-immunostained for Wnt8 and NAH46 epitope ( N- acetyl HS) or HepSS-1 epitope ( N- sulfo HS) at the VMZ ( f , i ). Signal intensities along white arrows were plotted ( g , j ), starting and ending points as indicated by yellow and cyan arrowheads, respectively ( f , i ). Scatter plots show the indicated signal intensities for every pixel along the arrow, presented with correlation coefficients ( r ) ( h , k ). Note N- sulfo-rich HS clusters inside cells, with Wnt8 (orange arrowheads) and without Wnt8 (white arrowheads). Staining of N- acetyl HS was absent from the Wnt8 puncta inside cells (open arrowheads in f ). Images are a representative of at least two independent experiments. Amounts of mRNA (ng/embryo): mV-wnt8 , 1.0; mRFP , 0.50. Scale bars, 100 μm ( a ); 10 μm ( c – f , i ). a.u., arbitrary units
Figure Legend Snippet: Endogenous Wnt8 shows not only a gradient but also HS-dependent punctate distributions. a The gradient of the endogenous Wnt8 protein from the lateral to mid-dorsal marginal zones at the mid-gastrula stage (st. 11.5). The observed region is indicated by the cyan box (reported localization of wnt8 mRNA in magenta). Embryos were flat-mounted under a coverslip and the image was acquired using automatic tiling with the maximum intensity projection of z -stacks (junctions of tiling appeared darker, arrowheads). b Quantification of the gradient of Wnt8 staining ( a ). The junctions of tiling are indicated by dotted lines, showing the drop in intensity. c A high-resolution image of endogenous Wnt8 staining in the VMZ. wnt8 is expressed in the underlying mesoderm, but not in the superficial layer in Xenopus embryos. An optical section at the subapical region of cells (basal to the tight junction) of the superficial layer is shown (see also Supplementary Fig. 2b, d ). d Exogenous mV-Wnt8 expression and heparitinase treatment. Experimental procedures are illustrated on the left. Embryos were observed at stage 10.5. The source cells are indicated with “*”. e Immunostaining of HS chains with NAH46 (for N- acetyl HS) or HepSS-1 (for N- sulfo HS). Notably HepSS-1 staining shows puncta inside cells (orange arrowheads). f – k The colocalization of endogenous Wnt8 and a subpopulation of HS. Gastrula embryos (st. 10.5) were co-immunostained for Wnt8 and NAH46 epitope ( N- acetyl HS) or HepSS-1 epitope ( N- sulfo HS) at the VMZ ( f , i ). Signal intensities along white arrows were plotted ( g , j ), starting and ending points as indicated by yellow and cyan arrowheads, respectively ( f , i ). Scatter plots show the indicated signal intensities for every pixel along the arrow, presented with correlation coefficients ( r ) ( h , k ). Note N- sulfo-rich HS clusters inside cells, with Wnt8 (orange arrowheads) and without Wnt8 (white arrowheads). Staining of N- acetyl HS was absent from the Wnt8 puncta inside cells (open arrowheads in f ). Images are a representative of at least two independent experiments. Amounts of mRNA (ng/embryo): mV-wnt8 , 1.0; mRFP , 0.50. Scale bars, 100 μm ( a ); 10 μm ( c – f , i ). a.u., arbitrary units

Techniques Used: Staining, Expressing, Immunostaining

32) Product Images from "Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis"

Article Title: Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis

Journal: Journal of Clinical Investigation

doi:

Release and uptake of HBP. ( a ) Release of HBP from activated human neutrophils was followed after stimulating isolated PMNs with the indicated concentrations of PMA in the absence (gray bars) or presence (open bars) of HUVECs (ECs) cultured in 48-well plates. After removal of the cell suspension from the plate and centrifugation, the concentration of HBP in the cell-free supernatant was determined by ELISA. Experiments were done in triplicate; means ± SD are presented. ( b ) 35 S-labeled HBP binding sites were isolated from whole lysates of HUVECs by affinity chromatography on HBP-streptavidin agarose. Fractions eluted at 300, 400, or 500 mM NaCl were incubated in the presence (+) or absence (–) of C ABC (top) or HNO 2 (bottom). Cleavage products were separated on agarose gels and viewed by phosphoimaging. ( c ) Affinity-purified HBP binding sites eluted at 300 mM NaCl (left) or whole HUVEC lysates (right) were incubated in the absence (–) or presence (+) of C ABC and heparitinase. The cleavage products were separated by SDS-PAGE, electrotransferred to Zeta-Probe membranes, and probed by mAb 3G10. The relative positions of known endothelial proteoglycans of the heparan sulfate type are indicated by stars (from top to bottom): perlecan ( > 200 kDa), syndecan-3 (125 kDa), syndecan-1 (90 kDa), glypican (64 kDa), syndecan-2 (48 kDa), and syndecan-4 (35 kDa). The relative molecular masses of marker proteins are given on the left.
Figure Legend Snippet: Release and uptake of HBP. ( a ) Release of HBP from activated human neutrophils was followed after stimulating isolated PMNs with the indicated concentrations of PMA in the absence (gray bars) or presence (open bars) of HUVECs (ECs) cultured in 48-well plates. After removal of the cell suspension from the plate and centrifugation, the concentration of HBP in the cell-free supernatant was determined by ELISA. Experiments were done in triplicate; means ± SD are presented. ( b ) 35 S-labeled HBP binding sites were isolated from whole lysates of HUVECs by affinity chromatography on HBP-streptavidin agarose. Fractions eluted at 300, 400, or 500 mM NaCl were incubated in the presence (+) or absence (–) of C ABC (top) or HNO 2 (bottom). Cleavage products were separated on agarose gels and viewed by phosphoimaging. ( c ) Affinity-purified HBP binding sites eluted at 300 mM NaCl (left) or whole HUVEC lysates (right) were incubated in the absence (–) or presence (+) of C ABC and heparitinase. The cleavage products were separated by SDS-PAGE, electrotransferred to Zeta-Probe membranes, and probed by mAb 3G10. The relative positions of known endothelial proteoglycans of the heparan sulfate type are indicated by stars (from top to bottom): perlecan ( > 200 kDa), syndecan-3 (125 kDa), syndecan-1 (90 kDa), glypican (64 kDa), syndecan-2 (48 kDa), and syndecan-4 (35 kDa). The relative molecular masses of marker proteins are given on the left.

Techniques Used: Isolation, Cell Culture, Centrifugation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, Affinity Chromatography, Incubation, Affinity Purification, SDS Page, Marker

33) Product Images from "A strategy for rapid sequencing of heparan sulfate and heparin saccharides"

Article Title: A strategy for rapid sequencing of heparan sulfate and heparin saccharides

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

doi:

Purification and IGS of an HS decasaccharide. ( A ) SAX-HPLC of a pool of HS decasaccharides derived by heparitinase treatment of porcine mucosal HS. The arrow indicates the peak selected for sequencing. ( B ) IGS of the purified HS decasaccharide on a 16-cm 33% Tris-acetate gel with the combinations of partial HNO 2 and exoenzyme treatments indicated (lane 1, untreated, 20 pmol; other lanes correspond to ≈400 pmol per lane of starting sample for partial HNO 2 digest). ( C ) Determining the sequence of the nonreducing disaccharide unit of the HS decasaccharide with the mercuric acetate (MA) and G6Sase treatments shown (lane 1, untreated; ≈40 pmol per lane).
Figure Legend Snippet: Purification and IGS of an HS decasaccharide. ( A ) SAX-HPLC of a pool of HS decasaccharides derived by heparitinase treatment of porcine mucosal HS. The arrow indicates the peak selected for sequencing. ( B ) IGS of the purified HS decasaccharide on a 16-cm 33% Tris-acetate gel with the combinations of partial HNO 2 and exoenzyme treatments indicated (lane 1, untreated, 20 pmol; other lanes correspond to ≈400 pmol per lane of starting sample for partial HNO 2 digest). ( C ) Determining the sequence of the nonreducing disaccharide unit of the HS decasaccharide with the mercuric acetate (MA) and G6Sase treatments shown (lane 1, untreated; ≈40 pmol per lane).

Techniques Used: Purification, High Performance Liquid Chromatography, Derivative Assay, Sequencing

34) Product Images from "Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1 *"

Article Title: Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.179663

CD47 is modified by heparan and chondroitin sulfate chains attached to Ser residues in the IgV domain. A , proteoglycans from Jurkat conditioned ( cond ) medium were digested in solution without enzymes ( 1st lane ), with 12.5 mIU/ml heparitinase ( Hep-ase
Figure Legend Snippet: CD47 is modified by heparan and chondroitin sulfate chains attached to Ser residues in the IgV domain. A , proteoglycans from Jurkat conditioned ( cond ) medium were digested in solution without enzymes ( 1st lane ), with 12.5 mIU/ml heparitinase ( Hep-ase

Techniques Used: Modification

35) Product Images from "2- and 6-O-sulfated proteoglycans have distinct and complementary roles in cranial axon guidance and motor neuron migration"

Article Title: 2- and 6-O-sulfated proteoglycans have distinct and complementary roles in cranial axon guidance and motor neuron migration

Journal: Development (Cambridge, England)

doi: 10.1242/dev.126854

Hs6st1 and Hs6st2 regulate cranial axon guidance, but not FBM neuron migration. (A) 11.5 dpc hindbrain explants were treated with inhibitors or implanted with beads containing growth factors. (B) Hindbrain explants ( n =4) were treated with vehicle or heparitinase and immunostained for ISL1. Asterisks indicate the midline. (C) Schematic representation of FBM neuron migration and ISH of 12.5 dpc hindbrains to reveal Hs6st1 and Hs6st2 expression relative to Isl1- positive migrating FBM neurons (VII m ) and the paired facial motor nuclei (VII n ). (D) Isl1 ISH of Hs6st1 −/− ( n =3), Hs6st2 −/− ( n =10) , Hs6st1 − / − ;Hs6st2 − / − (minor defects in 2/5 mutants examined) and wild-type ( n =6) hindbrains; the arrow indicates a minor defect in FBM neuron migration. (E) Lateral view of 11.5 dpc Hs6st1 −/− , Hs6st2 −/− and Hs6st1 −/− ;Hs6st2 −/− mutant ( n =3 each) and wild-type ( n =5) heads after immunolabelling for TUJ1. Green arrows indicate delayed extension of the mandibular (V md ) and maxillary (V mx ) nerves from the trigeminal ganglion (V g ), red arrows indicate abnormal chorda tympani (VII ct ) and facial branchiomotor nerve (VII bm ) branches from the facial ganglion (VII g ) and open triangles the lack of ophthalmic (V op ) and greater superficial petrosal (VII gspn ) nerves. Scale bars: 200 μm in B,C,E; 100 μm in D.
Figure Legend Snippet: Hs6st1 and Hs6st2 regulate cranial axon guidance, but not FBM neuron migration. (A) 11.5 dpc hindbrain explants were treated with inhibitors or implanted with beads containing growth factors. (B) Hindbrain explants ( n =4) were treated with vehicle or heparitinase and immunostained for ISL1. Asterisks indicate the midline. (C) Schematic representation of FBM neuron migration and ISH of 12.5 dpc hindbrains to reveal Hs6st1 and Hs6st2 expression relative to Isl1- positive migrating FBM neurons (VII m ) and the paired facial motor nuclei (VII n ). (D) Isl1 ISH of Hs6st1 −/− ( n =3), Hs6st2 −/− ( n =10) , Hs6st1 − / − ;Hs6st2 − / − (minor defects in 2/5 mutants examined) and wild-type ( n =6) hindbrains; the arrow indicates a minor defect in FBM neuron migration. (E) Lateral view of 11.5 dpc Hs6st1 −/− , Hs6st2 −/− and Hs6st1 −/− ;Hs6st2 −/− mutant ( n =3 each) and wild-type ( n =5) heads after immunolabelling for TUJ1. Green arrows indicate delayed extension of the mandibular (V md ) and maxillary (V mx ) nerves from the trigeminal ganglion (V g ), red arrows indicate abnormal chorda tympani (VII ct ) and facial branchiomotor nerve (VII bm ) branches from the facial ganglion (VII g ) and open triangles the lack of ophthalmic (V op ) and greater superficial petrosal (VII gspn ) nerves. Scale bars: 200 μm in B,C,E; 100 μm in D.

Techniques Used: Migration, In Situ Hybridization, Expressing, Mutagenesis

36) Product Images from "Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor"

Article Title: Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-10-5

Confocal microscopy illustration of samples of MCF-7 cells A-C) without treatment; D-F) treated 30 minutes with heparitinase (in figure F a group of three cells), Cells were stained with ErbB4 carboxy-terminus specific HFR-1 antibody . Accumulation of immunoreactive perinuclear granules is indicated by arrows. G) Activity of Heparitinase was controlled by simultaneous Western analysis of ErbB4 cleavage from parallel samples.
Figure Legend Snippet: Confocal microscopy illustration of samples of MCF-7 cells A-C) without treatment; D-F) treated 30 minutes with heparitinase (in figure F a group of three cells), Cells were stained with ErbB4 carboxy-terminus specific HFR-1 antibody . Accumulation of immunoreactive perinuclear granules is indicated by arrows. G) Activity of Heparitinase was controlled by simultaneous Western analysis of ErbB4 cleavage from parallel samples.

Techniques Used: Confocal Microscopy, Staining, Activity Assay, Western Blot

A) The effects of heparitinase and PMA can be reverted by adding 40 μM TAPI-0 to the incubation medium . MCF-7 cells were treated for 30 minutes and Triton-X100 soluble lysates containing 40 μg protein were subjected to immunoblot. The position of full length ErbB4 protein and ErbB4 80 kDa fragment as detected by the polyclonal sc-283 anti ErbB4 antibody are indicated by arrows. B) The effect of heparitinase is specific for TACE-cleavable (JM-a) ErbB4 isoform. MCF-7 cells were transiently transfected with ErbB4 JM-a CYT2HA or JM-b CYT2HA gene construct. Lysates were subjected to immunoblot with HA-specific monoclonal antibody. C) The effect of heparitinase treatment could be demonstrated in T47D cells treated similarly to MCF-7 cells. D) Degradation of chondroitin sulfate did not increase the formation of ErbB4 80 kDa fragment in T47D cells. E) Heat inactivated incubation medium from heparitinase treatment of T47D cells had only small effect on ErbB4 80 kDa fragment formation. The intensity of the ErbB4 80 kDa fragment staining as indicated in C and D was quantified with ImageJ software vs. 1.38 (NIH, USA). Beta-actin was used as load control (not shown). Abbreviations: Htase, heparitinase; Ctase, chondroitinase, PMA, phorbol myristyl acetate. Ht med. heat inactivated heparitinase incubation medium. The images are representative of at least three independent analyses.
Figure Legend Snippet: A) The effects of heparitinase and PMA can be reverted by adding 40 μM TAPI-0 to the incubation medium . MCF-7 cells were treated for 30 minutes and Triton-X100 soluble lysates containing 40 μg protein were subjected to immunoblot. The position of full length ErbB4 protein and ErbB4 80 kDa fragment as detected by the polyclonal sc-283 anti ErbB4 antibody are indicated by arrows. B) The effect of heparitinase is specific for TACE-cleavable (JM-a) ErbB4 isoform. MCF-7 cells were transiently transfected with ErbB4 JM-a CYT2HA or JM-b CYT2HA gene construct. Lysates were subjected to immunoblot with HA-specific monoclonal antibody. C) The effect of heparitinase treatment could be demonstrated in T47D cells treated similarly to MCF-7 cells. D) Degradation of chondroitin sulfate did not increase the formation of ErbB4 80 kDa fragment in T47D cells. E) Heat inactivated incubation medium from heparitinase treatment of T47D cells had only small effect on ErbB4 80 kDa fragment formation. The intensity of the ErbB4 80 kDa fragment staining as indicated in C and D was quantified with ImageJ software vs. 1.38 (NIH, USA). Beta-actin was used as load control (not shown). Abbreviations: Htase, heparitinase; Ctase, chondroitinase, PMA, phorbol myristyl acetate. Ht med. heat inactivated heparitinase incubation medium. The images are representative of at least three independent analyses.

Techniques Used: Incubation, Transfection, Construct, Staining, Software

Exogeneous heparin or HS inhibit heparitinase-induced ErbB4 80 kDa fragment formation . A) Small concentrations of heparin slightly enhance the heparitinase-induced ErbB4 80 kDa fragment formation, whereas 1 μg/ml heparin and higher concentrations inhibit ErbB4 80 kDa fragment formation as indicated in lysates of T47D cells. B) Incubation with increasing concentrations of bovine lung heparin only did not have marked effect on cells. N. s., non-specific staining in A and B. C) Both heparin and heparan sulfate inhibited activity of recombinant TACE at high concentrations but displayed some enhancement of enzyme activity at low concentrations D) Heparitinase treatment of living MCF-7 cells enhanced cleavage of fluorescent TACE substrate peptide (p = 6 × 10 -11 ) and the effect of heparitinase could be largely reverted by adding 40 μM TAPI-0 to the incubation medium (p = 10 -5 ). The p-values were calculated with two-tailed pairwise Student's test comparing all time points. The enzyme activity analysis was performed three times with similar results. The data shown represents results from a single assay.
Figure Legend Snippet: Exogeneous heparin or HS inhibit heparitinase-induced ErbB4 80 kDa fragment formation . A) Small concentrations of heparin slightly enhance the heparitinase-induced ErbB4 80 kDa fragment formation, whereas 1 μg/ml heparin and higher concentrations inhibit ErbB4 80 kDa fragment formation as indicated in lysates of T47D cells. B) Incubation with increasing concentrations of bovine lung heparin only did not have marked effect on cells. N. s., non-specific staining in A and B. C) Both heparin and heparan sulfate inhibited activity of recombinant TACE at high concentrations but displayed some enhancement of enzyme activity at low concentrations D) Heparitinase treatment of living MCF-7 cells enhanced cleavage of fluorescent TACE substrate peptide (p = 6 × 10 -11 ) and the effect of heparitinase could be largely reverted by adding 40 μM TAPI-0 to the incubation medium (p = 10 -5 ). The p-values were calculated with two-tailed pairwise Student's test comparing all time points. The enzyme activity analysis was performed three times with similar results. The data shown represents results from a single assay.

Techniques Used: Incubation, Staining, Activity Assay, Recombinant, Two Tailed Test

A) TACE antibody was shown immunoprecipitate Syndecan-4 shown as high molecular weight smear in immunoblot by anti Syndecan-4 . The co-immunoprecipitation was abolished by heparitinase treatment. B) Syndecan-4 and TACE colocalize in MCF-7 cells. The colocalization is disrupted by heparitinase treatment.
Figure Legend Snippet: A) TACE antibody was shown immunoprecipitate Syndecan-4 shown as high molecular weight smear in immunoblot by anti Syndecan-4 . The co-immunoprecipitation was abolished by heparitinase treatment. B) Syndecan-4 and TACE colocalize in MCF-7 cells. The colocalization is disrupted by heparitinase treatment.

Techniques Used: Molecular Weight, Immunoprecipitation

37) Product Images from "Glypican-3-mediated oncogenesis involves the Insulin-like growth factor-signaling pathway"

Article Title: Glypican-3-mediated oncogenesis involves the Insulin-like growth factor-signaling pathway

Journal: Carcinogenesis

doi: 10.1093/carcin/bgn091

GPC3 promotes cell growth in NIH3T3 cells. ( A ) Transient expression of GPC3 in HEK293T cells. Cells were transfected with pgpc3-GFP, and 48 h later, cytosolic extracts were analyzed by western blot using anti-GFP (lanes 1 and 2), 1G12 (lanes 3 and 4) or anti-CC3 (lanes 5 and 6). Molecular weight markers are indicated on the left. The bracket indicates glycanated GPC3 proteins; the arrow indicates the nascent GPC3 protein; the arrowhead indicates the 65 kDa core protein and the asterisk indicates the 40 kDa convertase-cleaved fragment. ( B ) Reverse transcription–polymerase chain reaction analysis of gpc3 mRNA in stable lines. Lane 1, the parental NIH3T3 cells; lane 2, vector control cells; lane 3, GPC3-60 cells; lane 4, GPC3-65 cells and lane 5, positive control (+). Mouse actin was served as the RNA loading control. ( C ) Expression of GPC3 protein in stable lines. Six hundred micrograms of cytosolic extracts were immunoprecipitated with anti-CC3, digested by heparitinase and then blotted with 1G12. Lane 1, the parental cells; lane 2, GPC3-60 cells and lane 3, GPC3-65 cells. The open arrow indicates the GPC3 core protein, and the black arrow indicates the antibody heavy chain. ( D ) Morphology of the cells. GPC3-60 and GPC3-65 cells revealed increased nucleus-to-cytoplasm (N/C) ratio and multinucleation in Papanicolaou stain ( a and b ). Parental and vector control cells were shown as a comparison ( c and d ). ( E ) Growth rate of the GPC3-expressing cells in serum-free medium. Cells were seeded in 35 mm plates in triplicate and cultured in Dulbecco's modified Eagle's medium without serum. Cells were harvested at 48 h intervals and were counted in triplicates. ( F ) Colony formation in soft agar was observed for GPC3-expressing line GPC3-60 and GPC3-65 (a and b), but not for the parental or vector control cells (c and d).
Figure Legend Snippet: GPC3 promotes cell growth in NIH3T3 cells. ( A ) Transient expression of GPC3 in HEK293T cells. Cells were transfected with pgpc3-GFP, and 48 h later, cytosolic extracts were analyzed by western blot using anti-GFP (lanes 1 and 2), 1G12 (lanes 3 and 4) or anti-CC3 (lanes 5 and 6). Molecular weight markers are indicated on the left. The bracket indicates glycanated GPC3 proteins; the arrow indicates the nascent GPC3 protein; the arrowhead indicates the 65 kDa core protein and the asterisk indicates the 40 kDa convertase-cleaved fragment. ( B ) Reverse transcription–polymerase chain reaction analysis of gpc3 mRNA in stable lines. Lane 1, the parental NIH3T3 cells; lane 2, vector control cells; lane 3, GPC3-60 cells; lane 4, GPC3-65 cells and lane 5, positive control (+). Mouse actin was served as the RNA loading control. ( C ) Expression of GPC3 protein in stable lines. Six hundred micrograms of cytosolic extracts were immunoprecipitated with anti-CC3, digested by heparitinase and then blotted with 1G12. Lane 1, the parental cells; lane 2, GPC3-60 cells and lane 3, GPC3-65 cells. The open arrow indicates the GPC3 core protein, and the black arrow indicates the antibody heavy chain. ( D ) Morphology of the cells. GPC3-60 and GPC3-65 cells revealed increased nucleus-to-cytoplasm (N/C) ratio and multinucleation in Papanicolaou stain ( a and b ). Parental and vector control cells were shown as a comparison ( c and d ). ( E ) Growth rate of the GPC3-expressing cells in serum-free medium. Cells were seeded in 35 mm plates in triplicate and cultured in Dulbecco's modified Eagle's medium without serum. Cells were harvested at 48 h intervals and were counted in triplicates. ( F ) Colony formation in soft agar was observed for GPC3-expressing line GPC3-60 and GPC3-65 (a and b), but not for the parental or vector control cells (c and d).

Techniques Used: Expressing, Transfection, Western Blot, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Immunoprecipitation, Papanicolaou Stain, Cell Culture, Modification

Related Articles

Fluorescence:

Article Title: Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface *
Article Snippet: .. Erythrocytes (2.5 × 105 ) were treated with heparitinase from Flavobacterium heparinum (EC 4.2.2.8; Seikagaku Corp., Tokyo, Japan) or neuraminidase from Vibrio cholerae (EC 3.2.1.18; Sigma) for 1 h at 37 °C, and then washed in fluorescence-activated cell sorting buffer (2% fetal calf serum and 0.1% NaN3 in phosphate-buffered saline). .. The enzyme-treated or -untreated cells (1 × 105 cells) were resuspended in 27 n m solutions of the purified recombinant proteins and incubated for 1 h at 4 °C.

Incubation:

Article Title: A homeostatic function of CXCR2 signalling in articular cartilage
Article Snippet: .. Hip caps were incubated overnight at 37°C either in phosphate buffered saline (PBS) or in PBS containing 5 mU/mL heparitinase (Seikagaku). .. Total proteins were precipitated from supernatants using trichloroacetic acid and assessed by western blotting.

Generated:

Article Title: Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line
Article Snippet: .. Biotinylated mouse anti-ΔHS (3G10) antibodies, recognizing HS neo-epitope, generated by the digestion with heparitinase I from Flavobacterium heparinum and heparitinase I (Flavobacterium heparinum ) were purchased from Seikagaku Corporation, (Tokyo, Japan). ..

Article Title: Heparan sulfate dependent signaling of fibroblast growth factor (FGF) 18 by chondrocyte-derived perlecan
Article Snippet: .. Antibodies against HS (10E4) and heparinase III generated HS-stubs (3G10) were purchased from Seikagaku Corp., Tokyo, Japan. .. Antibodies reactive for chondroitinase ABC generated unsulfated (1B5), 4-sulfated (2B6), 6-sulfated (3B3) CS-stubs and KS (5D4) were provided by Prof. Bruce Caterson, Cardiff University, Cardiff, Wales, UK.

other:

Article Title: Polyamines release the let-7b-mediated suppression of initiation codon recognition during the protein synthesis of EXT2
Article Snippet: Chondroitinase ABC (ChaseABC) from Proteus vulgaris , chondroitinase ACII (ChaseACII) from Arthrobacter aurescens , Heparinase I, heparinase II, heparinase III from Flavobacterium heparinum , unsaturated disaccharides of CS (ΔDi-0S, ΔDi-4S, ΔDi-6S, ΔDiUA-2S, ΔDi-diSE , ΔDi-diSB , ΔDi-diSD , ΔDi-TriS), and of HS (ΔDi-0SHS , ΔDi-NS, ΔDi-6SHS , ΔDi-NS6S ΔDi-NSUA2S, ΔDi-TriSHS ) were purchased from Seikagaku Corp., Tokyo, Japan.

Article Title: Distinct effects of glucose and glucosamine on vascular endothelial and smooth muscle cells: Evidence for a protective role for glucosamine in atherosclerosis
Article Snippet: Heparinase, heparitinase and chondroitinase ABC were purchased from Seikagaku America Inc (Bethesda, MD).

Modification:

Article Title: Effects of Modifiers of Glycosaminoglycan Biosynthesis on Outflow Facility in Perfusion Culture
Article Snippet: .. Draq5 was obtained from Axxora (San Diego, CA); normal goat serum, mounting medium (Fluoromount G), and chamber slides (Labtek II) from Fisher Scientific (Pittsburgh, PA); chondroitinase ABC protease-free (from Proteus vulgaris ) and heparitinase (from Flavobacterium heparinum ) from Seikagaku Corp. (Tokyo, Japan); hyaluronidase (from Streptomyces hyalurolyticus ) from ICN Biomedicals (Aurora, OH); Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin-amphotericin B from Invitrogen (Carlsbad, CA); fetal bovine serum (FBS) from HyClone (Logan, UT); protein markers (Precision Plus) from Bio-Rad (Hercules, CA); and anti-rabbit secondary antibody (IRDye680) from Licor Biosciences (Lincoln, NE). ..

FACS:

Article Title: Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface *
Article Snippet: .. Erythrocytes (2.5 × 105 ) were treated with heparitinase from Flavobacterium heparinum (EC 4.2.2.8; Seikagaku Corp., Tokyo, Japan) or neuraminidase from Vibrio cholerae (EC 3.2.1.18; Sigma) for 1 h at 37 °C, and then washed in fluorescence-activated cell sorting buffer (2% fetal calf serum and 0.1% NaN3 in phosphate-buffered saline). .. The enzyme-treated or -untreated cells (1 × 105 cells) were resuspended in 27 n m solutions of the purified recombinant proteins and incubated for 1 h at 4 °C.

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    Seikagaku heparitinase ii
    Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, <t>heparitinase</t> II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.
    Heparitinase Ii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.

    Journal: The Journal of Cell Biology

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

    doi:

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

    Article Snippet: Although trypsin treatment completely inhibited the reaction (Fig. , bottom right ), activated BAEC exposure to heparinase I, heparitinase II, or heparitinase III only had moderate inhibitory effects on L-selectin binding (Fig. , top right and middle ).

    Techniques: Immunofluorescence

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

    Journal: The Journal of Cell Biology

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

    doi:

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

    Article Snippet: Although trypsin treatment completely inhibited the reaction (Fig. , bottom right ), activated BAEC exposure to heparinase I, heparitinase II, or heparitinase III only had moderate inhibitory effects on L-selectin binding (Fig. , top right and middle ).

    Techniques: Immunofluorescence, Staining

    Recombinant BAEBL/Fc binds to erythrocytes via HS and sialic acid. A , binding inhibition of BAEBL/Fc to enzyme-treated erythrocytes. Erythrocytes (10 5 cells) were pretreated with buffer; 0.0016, 0.016, 0.16, or 1.6 milliunits of heparitinase; or 0.004,

    Journal:

    Article Title: Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface *

    doi: 10.1074/jbc.M109.021576

    Figure Lengend Snippet: Recombinant BAEBL/Fc binds to erythrocytes via HS and sialic acid. A , binding inhibition of BAEBL/Fc to enzyme-treated erythrocytes. Erythrocytes (10 5 cells) were pretreated with buffer; 0.0016, 0.016, 0.16, or 1.6 milliunits of heparitinase; or 0.004,

    Article Snippet: Erythrocytes (2.5 × 105 ) were treated with heparitinase from Flavobacterium heparinum (EC 4.2.2.8; Seikagaku Corp., Tokyo, Japan) or neuraminidase from Vibrio cholerae (EC 3.2.1.18; Sigma) for 1 h at 37 °C, and then washed in fluorescence-activated cell sorting buffer (2% fetal calf serum and 0.1% NaN3 in phosphate-buffered saline).

    Techniques: Recombinant, Binding Assay, Inhibition

    HS-dependent binding is involved in merozoite invasion. A , inhibition of merozoite invasion of enzyme-treated erythrocytes. Erythrocytes (10 7 cells) were treated with buffer, with 0.016, 0.16, 1.6, or 16 milliunits ( mU ) of heparitinase, or with 0.04,

    Journal:

    Article Title: Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface *

    doi: 10.1074/jbc.M109.021576

    Figure Lengend Snippet: HS-dependent binding is involved in merozoite invasion. A , inhibition of merozoite invasion of enzyme-treated erythrocytes. Erythrocytes (10 7 cells) were treated with buffer, with 0.016, 0.16, 1.6, or 16 milliunits ( mU ) of heparitinase, or with 0.04,

    Article Snippet: Erythrocytes (2.5 × 105 ) were treated with heparitinase from Flavobacterium heparinum (EC 4.2.2.8; Seikagaku Corp., Tokyo, Japan) or neuraminidase from Vibrio cholerae (EC 3.2.1.18; Sigma) for 1 h at 37 °C, and then washed in fluorescence-activated cell sorting buffer (2% fetal calf serum and 0.1% NaN3 in phosphate-buffered saline).

    Techniques: Binding Assay, Inhibition

    Complexes formed between perlecan, FGF18 and FGFR3 or perlecan and FGFR3. Perlecans were adsorbed onto wells of a 96 well plate and were either undigested or digested in situ with heparinase III, mammalian heparanase or chondroitinase ABC before performing

    Journal: Biochemistry

    Article Title: Heparan sulfate dependent signaling of fibroblast growth factor (FGF) 18 by chondrocyte-derived perlecan

    doi: 10.1021/bi1005199

    Figure Lengend Snippet: Complexes formed between perlecan, FGF18 and FGFR3 or perlecan and FGFR3. Perlecans were adsorbed onto wells of a 96 well plate and were either undigested or digested in situ with heparinase III, mammalian heparanase or chondroitinase ABC before performing

    Article Snippet: Antibodies against HS (10E4) and heparinase III generated HS-stubs (3G10) were purchased from Seikagaku Corp., Tokyo, Japan.

    Techniques: In Situ

    Ternary complexes formed between perlecan, either FGF2 or FGF18 and either FGF receptor type 1 or 3. Perlecans were adsorbed onto wells of a 96 well plate and were either undigested or digested in situ with heparinase III, mammalian heparanase or chondroitinase

    Journal: Biochemistry

    Article Title: Heparan sulfate dependent signaling of fibroblast growth factor (FGF) 18 by chondrocyte-derived perlecan

    doi: 10.1021/bi1005199

    Figure Lengend Snippet: Ternary complexes formed between perlecan, either FGF2 or FGF18 and either FGF receptor type 1 or 3. Perlecans were adsorbed onto wells of a 96 well plate and were either undigested or digested in situ with heparinase III, mammalian heparanase or chondroitinase

    Article Snippet: Antibodies against HS (10E4) and heparinase III generated HS-stubs (3G10) were purchased from Seikagaku Corp., Tokyo, Japan.

    Techniques: In Situ