Structured Review

BioMarin heparitinase
<t>Heparitinase</t> treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p
Heparitinase, supplied by BioMarin, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization"

Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E10-04-0348

Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p
Figure Legend Snippet: Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

Techniques Used: Cell Culture, Activation Assay, Recombinant

BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p
Figure Legend Snippet: BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

Techniques Used: Binding Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Stable Transfection, Labeling, Plasmid Preparation

Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).
Figure Legend Snippet: Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

Techniques Used: Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page, Molecular Weight

A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p
Figure Legend Snippet: A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

Techniques Used: Binding Assay, Variant Assay, Activation Assay, Labeling

A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p
Figure Legend Snippet: A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

Techniques Used: Binding Assay, Variant Assay, Labeling, Activation Assay, Sample Prep, Positive Control

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    BioMarin heparitinase
    <t>Heparitinase</t> treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p
    Heparitinase, supplied by BioMarin, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/BioMarin
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-09
    91/100 stars
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    Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: Heparitinase treatment and blockade of sulfation diminish responses of cultured cells to BMP2. (A) Smad phosphorylation and p38 MAPK activation in mouse C2C12 and rat PC12 cells. Cells maintained in serum-free medium were treated with human recombinant BMP2 at 5 ng/ml for 1 h. Where indicated, cultures also were treated with 25 mIU/ml heparitinase 1 h before BMP addition and throughout the remainder of the assay. Cells lysates were subjected to immunoblotting for phospho-Smad1/5/8 (P-Smad) and active p38. Total Smad, total p38, and β-tubulin served as loading controls. (B and C) Kinetic profiles of BMP-induced Smad phosphorylation. C2C12 (B) or PC12 cells (C) were treated with heparitinase for 1 h, and BMP2 (5 ng/ml) was added. Cell lysates were collected at indicated time points and subjected to immunoblotting for P-Smad. Data are normalized to loading controls. (D and E) Exogenous heparin does not rescue cells from the effect of heparitinase treatment. C2C12 cells were treated with heparitinase for 1 h, and BMP2 (5 ng/ml), or BMP2 and heparin (3–100 μg/ml) were added for a subsequent hour. Cell lysates were subjected to immunoblotting for active p38 (D) or P-Smad (E), and band intensities were quantified. Data are from duplicate cultures for each condition and are normalized to loading controls. Both activation of p38 and Smad in response to BMP2 were substantially lower in heparitinase-treated cells than in untreated cells (*p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Cell Culture, Activation Assay, Recombinant

    BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: BMP binding to type II, but not type I, receptor subunits depends on HS. (A) Binding of 125 I-BMP4 to BMPRIA. C2C12 cells were transiently transfected with HA-tagged BMPRIA, treated with heparitinase (1 h at 37°C), incubated with 125 I-BMP4 (20 ng/ml, 2 h at room temperature), and cross-linked with BS 3 . Lysates were immunoprecipitated with anti-HA antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMP dimer (36 kDa, arrow 2), and cross-linked BMPRIA-BMP complexes (78 kDa; arrow 3) are shown. An arrow marked RI shows the location of uncrosslinked HA-tagged BMPRIA (60 kDa; as determined separately by immunoblotting). (B) Binding of 125 I-BMP4 to BMPRII. C2C12 cells were transfected stably with myc-tagged BMPRII (lanes labeled RII), or stably with BMPRII-myc and transiently with BMPRIA-HA (lanes labeled RI RII). Mock-transfected cells were transiently transfected with vector (pcDNA3.1) only. 125 I-BMP4 binding was carried out as in A. Lysates were immunoprecipitated with anti-myc antibodies, and precipitates subjected to SDS-PAGE and autoradiography. Locations of molecular sizes corresponding to BMP monomer (18 kDa; arrow 1), cross-linked BMPRIA-BMP complexes (78 kDa, arrow 2), cross-linked BMPRII-BMP complexes (93 kDa, arrow 3), and higher order complexes (∼153 kDa, arrow 4) are shown. Arrows RI and RII mark the locations of uncrosslinked BMPRIA-HA receptor (60 kDa) and uncrosslinked BMPRII-myc (75 kDa), as determined separately by immunoblotting. (C and D) Quantification of B. Results from cells transfected with BMPRII alone are shown in C, whereas those from cells transfected with both BMPRII and BMPRIA are in D (data are mean values ± SD of band intensities). Black bars quantify binding to cells not treated with heparitinase, whereas gray bars quantify binding to heparitinase-treated cells. The categories RI-BMP, RII-BMP, and higher-order complexes refer to the intensities of bands at arrows 2, 3, and 4, respectively, in B. Statistical significance of heparitinase effects was calculated by t test (*p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Binding Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Stable Transfection, Labeling, Plasmid Preparation

    Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: Assembly of heteromeric receptor complexes is HS-dependent. (A) C2C12 cells stably expressing BMPRII-myc were transiently transfected with BMPRIA-HA. After treatment with or without heparitinase for 1 h, BMP2 (10 ng/ml) was added for 2 h at room temperature and cross-linked for 30 min with BS 3 . Cell lysates were immunoprecipitated with anti-HA antibodies and precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc antibodies. The arrow shows the location of BMPRII-myc (75 kDa), as readily visualized in the blot of total cell lysates. (B) Long exposure of the blot in panel A. Arrow 1 shows the location of BMPRII. Arrow 2 marks bands with molecular weight corresponding to cross-linked BMPRII-BMP complexes (93 kDa). Larger bands, consistent with complexes containing BMPRI and BMPRII are also indicated (bracket 3).

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page, Molecular Weight

    A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: A non-heparin binding BMP2 variant is partially resistant to chlorate treatment. (A) Smad phosphorylation and p38 activation were assessed by immunoblotting in C2C12 and PC12 cells pretreated with various concentrations of chlorate (as indicated) for 48 h, and stimulated with either BMP2 (labeled B) or EHBMP2 (labeled E), at 5 ng/ml for 1 h. Total Smad, total p38 and β-tubulin served as loading controls. The data (mean values normalized to loading controls ± SD) are quantified in panels B (P-Smad in C2C12 cells), C (P-Smad in PC12 cells) and D (active p38 in PC12 cells). Effects of heparitinase were statistically significant for both BMP2 (black bars) and EHBMP2 (gray bars), but weaker for EHBMP2, particularly when cells were treated with intermediate chlorate levels (*, # p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Binding Assay, Variant Assay, Activation Assay, Labeling

    A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

    Journal: Molecular Biology of the Cell

    Article Title: Heparan Sulfate Acts as a Bone Morphogenetic Protein Coreceptor by Facilitating Ligand-induced Receptor Hetero-oligomerization

    doi: 10.1091/mbc.E10-04-0348

    Figure Lengend Snippet: A non-heparin binding BMP2 variant is resistant to heparitinase treatment. (A) Comparison of the N-terminal sequences of BMP2 and the engineered variant EH-BMP2, in which the first 12 amino acids have been replaced ( Ruppert et al. , 1996 ). Cationic residues in BMP2 are labeled with dots. (B) BMP2 and EHBMP2 are equal in potency. C2C12 cells were stimulated for 1 h with either BMP2 (circles) or EHBMP2 (triangles) at the indicated concentrations, lysed and subjected to immunoblotting for P-Smad. (C–E) Effect of heparitinase on p38 activation and Smad phosphorylation in BMP2- or EHBMP2-stimulated C2C12 cells. Cells were treated with heparitinase for 1 h and stimulated with either BMP2 or EHBMP2 for 1 h before sample preparation. Cell lysates were subjected to immunoblotting (panel C) for active p38 or P-Smad, with total p38 and β-tubulin serving as loading controls, and osmotic shock (sorbitol; 250 mM) as a positive control for active p38 (lane labeled “PC”). Band intensities were quantified and normalized to loading controls. In D and E, these data are plotted as mean values ± SD for each of the duplicate determinations shown in C. Significant effects of heparitinase on p38 activation and Smad phosphorylation are seen for BMP2-treated cells (*p

    Article Snippet: Heparitinase was a gift from BioMarin Pharmaceuticals (Novato, CA).

    Techniques: Binding Assay, Variant Assay, Labeling, Activation Assay, Sample Prep, Positive Control