human gdf5 rhgdf5 Search Results


human gdf5 rhgdf5  (R&D Systems)
92
R&D Systems human gdf5 rhgdf5
( A ) Primary murine chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with or without (–) 1 × 10 –8 M 1,25D prior to incubation with (+) or without (–) 100 ng/mL <t>rhGDF5</t> (for 30 minutes) and subjected to Western blot analyses for p-SMAD1/5/9, SMAD1, and β-actin. Data are representative of those obtained from 3 independent experiments. ( B ) Primary murine chondrocytes were pretreated for 18 hours with 1 × 10 –8 M 1,25D followed by treatment with 200 ng/mL rhGDF5 (for 4 hours). Gene expression analyses were performed for BMP and IHH target genes. Data are representative of those obtained from 5–7 independent experiments. One-way ANOVA followed by Fisher’s least significant difference test was used to analyze significance between all genotype groups. * P < 0.05 versus WT; # P < 0.05 versus rhGDF5; a indicates P < 0.05 versus rhGDF5 plus 1,25D. ( C ) IHC for VDR was performed on P7, P14, P30, and P60 entheses from WT, Hyp , and C –/– mice. In all representative pictures, the enthesis region is outlined with a black box. Scale bar: 20 μm. Data are representative of 6 mice per age or genotype group.
Human Gdf5 Rhgdf5, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant human gdf-5 protein, cf  (Bio-Techne corporation)
93
Bio-Techne corporation recombinant human gdf-5 protein, cf
( A ) Primary murine chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with or without (–) 1 × 10 –8 M 1,25D prior to incubation with (+) or without (–) 100 ng/mL <t>rhGDF5</t> (for 30 minutes) and subjected to Western blot analyses for p-SMAD1/5/9, SMAD1, and β-actin. Data are representative of those obtained from 3 independent experiments. ( B ) Primary murine chondrocytes were pretreated for 18 hours with 1 × 10 –8 M 1,25D followed by treatment with 200 ng/mL rhGDF5 (for 4 hours). Gene expression analyses were performed for BMP and IHH target genes. Data are representative of those obtained from 5–7 independent experiments. One-way ANOVA followed by Fisher’s least significant difference test was used to analyze significance between all genotype groups. * P < 0.05 versus WT; # P < 0.05 versus rhGDF5; a indicates P < 0.05 versus rhGDF5 plus 1,25D. ( C ) IHC for VDR was performed on P7, P14, P30, and P60 entheses from WT, Hyp , and C –/– mice. In all representative pictures, the enthesis region is outlined with a black box. Scale bar: 20 μm. Data are representative of 6 mice per age or genotype group.
Recombinant Human Gdf 5 Protein, Cf, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rhgdf5 w414r  (Biopharm GmbH)
90
Biopharm GmbH rhgdf5 w414r
A : 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5 <t>W414R</t> ), violet (GDF5 R399C ) and orange (GDF5 E491K ). The image of the GDF5 structure was visualized using PyMol ( http://www.pymol.org/ ). B : Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5 W414R , GDF5 R399C and GDF5 E491K . Note that GDF5 W414R and GDF5 E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.
Rhgdf5 W414r, supplied by Biopharm GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant human (rh) gdf5  (Biopharm GmbH)
90
Biopharm GmbH recombinant human (rh) gdf5
Clinical features of the affected family members with mutations in <t> GDF5 </t> .
Recombinant Human (Rh) Gdf5, supplied by Biopharm GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/recombinant human (rh) gdf5/product/Biopharm GmbH
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recombinant human growth  (Abcam)
92
Abcam recombinant human growth
Clinical features of the affected family members with mutations in <t> GDF5 </t> .
Recombinant Human Growth, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


( A ) Primary murine chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with or without (–) 1 × 10 –8 M 1,25D prior to incubation with (+) or without (–) 100 ng/mL rhGDF5 (for 30 minutes) and subjected to Western blot analyses for p-SMAD1/5/9, SMAD1, and β-actin. Data are representative of those obtained from 3 independent experiments. ( B ) Primary murine chondrocytes were pretreated for 18 hours with 1 × 10 –8 M 1,25D followed by treatment with 200 ng/mL rhGDF5 (for 4 hours). Gene expression analyses were performed for BMP and IHH target genes. Data are representative of those obtained from 5–7 independent experiments. One-way ANOVA followed by Fisher’s least significant difference test was used to analyze significance between all genotype groups. * P < 0.05 versus WT; # P < 0.05 versus rhGDF5; a indicates P < 0.05 versus rhGDF5 plus 1,25D. ( C ) IHC for VDR was performed on P7, P14, P30, and P60 entheses from WT, Hyp , and C –/– mice. In all representative pictures, the enthesis region is outlined with a black box. Scale bar: 20 μm. Data are representative of 6 mice per age or genotype group.

Journal: JCI Insight

Article Title: Impaired 1,25-dihydroxyvitamin D 3 action underlies enthesopathy development in the Hyp mouse model of X-linked hypophosphatemia

doi: 10.1172/jci.insight.163259

Figure Lengend Snippet: ( A ) Primary murine chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with or without (–) 1 × 10 –8 M 1,25D prior to incubation with (+) or without (–) 100 ng/mL rhGDF5 (for 30 minutes) and subjected to Western blot analyses for p-SMAD1/5/9, SMAD1, and β-actin. Data are representative of those obtained from 3 independent experiments. ( B ) Primary murine chondrocytes were pretreated for 18 hours with 1 × 10 –8 M 1,25D followed by treatment with 200 ng/mL rhGDF5 (for 4 hours). Gene expression analyses were performed for BMP and IHH target genes. Data are representative of those obtained from 5–7 independent experiments. One-way ANOVA followed by Fisher’s least significant difference test was used to analyze significance between all genotype groups. * P < 0.05 versus WT; # P < 0.05 versus rhGDF5; a indicates P < 0.05 versus rhGDF5 plus 1,25D. ( C ) IHC for VDR was performed on P7, P14, P30, and P60 entheses from WT, Hyp , and C –/– mice. In all representative pictures, the enthesis region is outlined with a black box. Scale bar: 20 μm. Data are representative of 6 mice per age or genotype group.

Article Snippet: For Western blot analyses, chondrocytes were pretreated for 0.5, 1, 4, or 18 hours with 1 × 10 –8 M 1,25D prior to exposure to recombinant human GDF5 (rhGDF5) (100 ng/mL; R&D Systems) for 30 minutes.

Techniques: Incubation, Western Blot, Expressing

A : 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5 W414R ), violet (GDF5 R399C ) and orange (GDF5 E491K ). The image of the GDF5 structure was visualized using PyMol ( http://www.pymol.org/ ). B : Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5 W414R , GDF5 R399C and GDF5 E491K . Note that GDF5 W414R and GDF5 E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: A : 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5 W414R ), violet (GDF5 R399C ) and orange (GDF5 E491K ). The image of the GDF5 structure was visualized using PyMol ( http://www.pymol.org/ ). B : Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5 W414R , GDF5 R399C and GDF5 E491K . Note that GDF5 W414R and GDF5 E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Sequencing, Binding Assay

Chicken micromass cells were infected with RCASBP(A) containing the coding sequence (cds) of either wild type GDF5 or the GDF5 variants GDF5 W414R , GDF5 R399C or GDF5 E491K . RCASBP(B) contained the cds of NOG and was used for co-transfection. Chicken micromass cultures and quantification of Alcian blue incorporation at 595 nm into the extracellular matrix (ECM) are shown for day 5. In the chicken micromass system, wild type GDF5 strongly induced chondrogenesis compared to the untransfected control. Chondrogenic differentiation was completely blocked in both, the control and wild type GDF5 cultures, when NOG is co-transfected. A similar pattern was observed for GDF5 R399C . Contrary, GDF5 W414R and GDF5 E491K exhibited insensitivity towards the antagonist. Values represent the mean of triplicates and error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; ***p≤0.001).

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Chicken micromass cells were infected with RCASBP(A) containing the coding sequence (cds) of either wild type GDF5 or the GDF5 variants GDF5 W414R , GDF5 R399C or GDF5 E491K . RCASBP(B) contained the cds of NOG and was used for co-transfection. Chicken micromass cultures and quantification of Alcian blue incorporation at 595 nm into the extracellular matrix (ECM) are shown for day 5. In the chicken micromass system, wild type GDF5 strongly induced chondrogenesis compared to the untransfected control. Chondrogenic differentiation was completely blocked in both, the control and wild type GDF5 cultures, when NOG is co-transfected. A similar pattern was observed for GDF5 R399C . Contrary, GDF5 W414R and GDF5 E491K exhibited insensitivity towards the antagonist. Values represent the mean of triplicates and error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; ***p≤0.001).

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Infection, Sequencing, Cotransfection, Transfection, Standard Deviation, Two Tailed Test

Binding affinities of GDF5 W414R to immobilized receptor ectodomains.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Binding affinities of GDF5 W414R to immobilized receptor ectodomains.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Binding Assay

NIH/3T3 cells were transfected with the BMP type I receptors, Bmpr1a or Bmpr1b, as well as with wild type GDF5 and the GDF5 variants GDF5 W414R , GDF5 R399C and GDF5 E491K . As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. A : No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5 E491K , whereas in case of GDF5 W414R and GDF5 R399C signaling activity was absent. B : Bmpr1a co-expression increased the signaling activity of wild type GDF5 and GDF5 E491K ; however, GDF5 W414R and GDF5 R399C were not able to induce reporter gene expression. C : Co-expression of Bmpr1b further increased the signaling activity of wild type GDF5 and GDF5 E491K compared to co-expression with Bmpr1a . In case of GDF5 W414R and GDF5 R399C , Bmpr1b co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: NIH/3T3 cells were transfected with the BMP type I receptors, Bmpr1a or Bmpr1b, as well as with wild type GDF5 and the GDF5 variants GDF5 W414R , GDF5 R399C and GDF5 E491K . As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. A : No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5 E491K , whereas in case of GDF5 W414R and GDF5 R399C signaling activity was absent. B : Bmpr1a co-expression increased the signaling activity of wild type GDF5 and GDF5 E491K ; however, GDF5 W414R and GDF5 R399C were not able to induce reporter gene expression. C : Co-expression of Bmpr1b further increased the signaling activity of wild type GDF5 and GDF5 E491K compared to co-expression with Bmpr1a . In case of GDF5 W414R and GDF5 R399C , Bmpr1b co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Transfection, Binding Assay, Luciferase, Activation Assay, Activity Assay, Expressing, Standard Deviation, Two Tailed Test

Bmpr1b wild type ( Bmpr1b +/+ ), heterozygous ( Bmpr1b +/− ) and homozygous ( Bmpr1b −/− ) mouse mesenchymal limb bud cells (E13.5) were stimulated with 5 nM recombinant human GDF5 protein (wild type GDF5 and GDF5 W414R ). Alcian blue incorporation into the extracellular matrix (ECM) was measured at 595 nm after five days of cultivation and four days of stimulation. A : Alcian blue staining of Bmpr1b +/+ cells exhibited a strong induction of chondrogenesis upon stimulation with both recombinant GDF5 proteins. B : Stimulation of Bmpr1b +/− cells resulted in a reduced chondrogenic activity of GDF5 W414R compared to wild type GDF5. C : In case of Bmpr1b knockout cells, stimulation with GDF5 W414R resulted in a complete loss of chondrogenic activity, compared to wild type GDF5. Values represent the mean of three replicates, error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (*p≤0.05; ***p≤0.001).

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Bmpr1b wild type ( Bmpr1b +/+ ), heterozygous ( Bmpr1b +/− ) and homozygous ( Bmpr1b −/− ) mouse mesenchymal limb bud cells (E13.5) were stimulated with 5 nM recombinant human GDF5 protein (wild type GDF5 and GDF5 W414R ). Alcian blue incorporation into the extracellular matrix (ECM) was measured at 595 nm after five days of cultivation and four days of stimulation. A : Alcian blue staining of Bmpr1b +/+ cells exhibited a strong induction of chondrogenesis upon stimulation with both recombinant GDF5 proteins. B : Stimulation of Bmpr1b +/− cells resulted in a reduced chondrogenic activity of GDF5 W414R compared to wild type GDF5. C : In case of Bmpr1b knockout cells, stimulation with GDF5 W414R resulted in a complete loss of chondrogenic activity, compared to wild type GDF5. Values represent the mean of three replicates, error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (*p≤0.05; ***p≤0.001).

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Recombinant, Staining, Activity Assay, Knock-Out, Standard Deviation, Two Tailed Test

Clinical features of the affected family members with mutations in GDF5 .

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Clinical features of the affected family members with mutations in GDF5 .

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques:

A : 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5 W414R ), violet (GDF5 R399C ) and orange (GDF5 E491K ). The image of the GDF5 structure was visualized using PyMol ( http://www.pymol.org/ ). B : Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5 W414R , GDF5 R399C and GDF5 E491K . Note that GDF5 W414R and GDF5 E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: A : 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5 W414R ), violet (GDF5 R399C ) and orange (GDF5 E491K ). The image of the GDF5 structure was visualized using PyMol ( http://www.pymol.org/ ). B : Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5 W414R , GDF5 R399C and GDF5 E491K . Note that GDF5 W414R and GDF5 E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Sequencing, Binding Assay

Chicken micromass cells were infected with RCASBP(A) containing the coding sequence (cds) of either wild type GDF5 or the GDF5 variants GDF5 W414R , GDF5 R399C or GDF5 E491K . RCASBP(B) contained the cds of NOG and was used for co-transfection. Chicken micromass cultures and quantification of Alcian blue incorporation at 595 nm into the extracellular matrix (ECM) are shown for day 5. In the chicken micromass system, wild type GDF5 strongly induced chondrogenesis compared to the untransfected control. Chondrogenic differentiation was completely blocked in both, the control and wild type GDF5 cultures, when NOG is co-transfected. A similar pattern was observed for GDF5 R399C . Contrary, GDF5 W414R and GDF5 E491K exhibited insensitivity towards the antagonist. Values represent the mean of triplicates and error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; ***p≤0.001).

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Chicken micromass cells were infected with RCASBP(A) containing the coding sequence (cds) of either wild type GDF5 or the GDF5 variants GDF5 W414R , GDF5 R399C or GDF5 E491K . RCASBP(B) contained the cds of NOG and was used for co-transfection. Chicken micromass cultures and quantification of Alcian blue incorporation at 595 nm into the extracellular matrix (ECM) are shown for day 5. In the chicken micromass system, wild type GDF5 strongly induced chondrogenesis compared to the untransfected control. Chondrogenic differentiation was completely blocked in both, the control and wild type GDF5 cultures, when NOG is co-transfected. A similar pattern was observed for GDF5 R399C . Contrary, GDF5 W414R and GDF5 E491K exhibited insensitivity towards the antagonist. Values represent the mean of triplicates and error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; ***p≤0.001).

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Infection, Sequencing, Cotransfection, Control, Transfection, Standard Deviation, Two Tailed Test

Binding affinities of GDF5 W414R to immobilized receptor ectodomains.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Binding affinities of GDF5 W414R to immobilized receptor ectodomains.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Binding Assay

NIH/3T3 cells were transfected with the BMP type I receptors, Bmpr1a or Bmpr1b, as well as with wild type GDF5 and the GDF5 variants GDF5 W414R , GDF5 R399C and GDF5 E491K . As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. A : No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5 E491K , whereas in case of GDF5 W414R and GDF5 R399C signaling activity was absent. B : Bmpr1a co-expression increased the signaling activity of wild type GDF5 and GDF5 E491K ; however, GDF5 W414R and GDF5 R399C were not able to induce reporter gene expression. C : Co-expression of Bmpr1b further increased the signaling activity of wild type GDF5 and GDF5 E491K compared to co-expression with Bmpr1a . In case of GDF5 W414R and GDF5 R399C , Bmpr1b co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: NIH/3T3 cells were transfected with the BMP type I receptors, Bmpr1a or Bmpr1b, as well as with wild type GDF5 and the GDF5 variants GDF5 W414R , GDF5 R399C and GDF5 E491K . As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. A : No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5 E491K , whereas in case of GDF5 W414R and GDF5 R399C signaling activity was absent. B : Bmpr1a co-expression increased the signaling activity of wild type GDF5 and GDF5 E491K ; however, GDF5 W414R and GDF5 R399C were not able to induce reporter gene expression. C : Co-expression of Bmpr1b further increased the signaling activity of wild type GDF5 and GDF5 E491K compared to co-expression with Bmpr1a . In case of GDF5 W414R and GDF5 R399C , Bmpr1b co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Transfection, Binding Assay, Luciferase, Activation Assay, Activity Assay, Expressing, Gene Expression, Standard Deviation, Two Tailed Test

Bmpr1b wild type ( Bmpr1b +/+ ), heterozygous ( Bmpr1b +/− ) and homozygous ( Bmpr1b −/− ) mouse mesenchymal limb bud cells (E13.5) were stimulated with 5 nM recombinant human GDF5 protein (wild type GDF5 and GDF5 W414R ). Alcian blue incorporation into the extracellular matrix (ECM) was measured at 595 nm after five days of cultivation and four days of stimulation. A : Alcian blue staining of Bmpr1b +/+ cells exhibited a strong induction of chondrogenesis upon stimulation with both recombinant GDF5 proteins. B : Stimulation of Bmpr1b +/− cells resulted in a reduced chondrogenic activity of GDF5 W414R compared to wild type GDF5. C : In case of Bmpr1b knockout cells, stimulation with GDF5 W414R resulted in a complete loss of chondrogenic activity, compared to wild type GDF5. Values represent the mean of three replicates, error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (*p≤0.05; ***p≤0.001).

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Bmpr1b wild type ( Bmpr1b +/+ ), heterozygous ( Bmpr1b +/− ) and homozygous ( Bmpr1b −/− ) mouse mesenchymal limb bud cells (E13.5) were stimulated with 5 nM recombinant human GDF5 protein (wild type GDF5 and GDF5 W414R ). Alcian blue incorporation into the extracellular matrix (ECM) was measured at 595 nm after five days of cultivation and four days of stimulation. A : Alcian blue staining of Bmpr1b +/+ cells exhibited a strong induction of chondrogenesis upon stimulation with both recombinant GDF5 proteins. B : Stimulation of Bmpr1b +/− cells resulted in a reduced chondrogenic activity of GDF5 W414R compared to wild type GDF5. C : In case of Bmpr1b knockout cells, stimulation with GDF5 W414R resulted in a complete loss of chondrogenic activity, compared to wild type GDF5. Values represent the mean of three replicates, error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (*p≤0.05; ***p≤0.001).

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Recombinant, Staining, Activity Assay, Knock-Out, Standard Deviation, Two Tailed Test

Mouse embryo s with the C57BL/6 genetic background at embryonic stages 11.5 (A–H), 12.5 (A′–H′) and 13.5 (A″–H″) were labeled with probes of Gdf5 (A and E), Nog (B and F), Bmpr1a (C and G) or Bmpr1b (D and H) and signals are shown in red. Representatively, two sections of the coronal dorsal axis (A–D) and the autopod transversal axis (E–H) are depicted. The signal for Gdf5 strongly co-localizes with the Nog and Bmpr1b expression pattern, whereas Bmpr1a expression is in direct proximity in the surrounding epithelium and underlying mesenchyme.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: Mouse embryo s with the C57BL/6 genetic background at embryonic stages 11.5 (A–H), 12.5 (A′–H′) and 13.5 (A″–H″) were labeled with probes of Gdf5 (A and E), Nog (B and F), Bmpr1a (C and G) or Bmpr1b (D and H) and signals are shown in red. Representatively, two sections of the coronal dorsal axis (A–D) and the autopod transversal axis (E–H) are depicted. The signal for Gdf5 strongly co-localizes with the Nog and Bmpr1b expression pattern, whereas Bmpr1a expression is in direct proximity in the surrounding epithelium and underlying mesenchyme.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Labeling, Expressing

A : During normal limb development, dimeric GDF5 (light/dark grey rhomb) is antagonized by NOG (black framed clamp) and thus balanced within the GDF5 signaling network. Downstream signaling is mediated via heteromeric receptor complexes consisting of each of the BMP type I receptors (BMPR1A and BMPR1B) in complex with the BMP type II receptor (BMPR2). Wild type GDF5 binds BMPR1A with a weaker affinity compared to BMPR1B as indicated by thin and thick arrows and additionally by Biacore binding affinities (KD). B : Summary of altered interaction of GDF5 mutations resulting in specific phenotypes. SYNS2 is characterized by GDF5 gain of function mutations, leading to an insensitivity of GDF5 towards its extracellular antagonist NOG. In contrast, BDA1 is caused by GDF5 loss of function mutations, which result specifically in absent BMPR1A signaling.

Journal: PLoS Genetics

Article Title: A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

doi: 10.1371/journal.pgen.1003846

Figure Lengend Snippet: A : During normal limb development, dimeric GDF5 (light/dark grey rhomb) is antagonized by NOG (black framed clamp) and thus balanced within the GDF5 signaling network. Downstream signaling is mediated via heteromeric receptor complexes consisting of each of the BMP type I receptors (BMPR1A and BMPR1B) in complex with the BMP type II receptor (BMPR2). Wild type GDF5 binds BMPR1A with a weaker affinity compared to BMPR1B as indicated by thin and thick arrows and additionally by Biacore binding affinities (KD). B : Summary of altered interaction of GDF5 mutations resulting in specific phenotypes. SYNS2 is characterized by GDF5 gain of function mutations, leading to an insensitivity of GDF5 towards its extracellular antagonist NOG. In contrast, BDA1 is caused by GDF5 loss of function mutations, which result specifically in absent BMPR1A signaling.

Article Snippet: Recombinant human (rh) GDF5 and its variant rhGDF5 W414R were dissolved in 10 mM HCl and provided by Biopharm GmbH.

Techniques: Binding Assay