Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 2. Characterization of recombinant GDF6, GDF7, and BMP10. A, immuno- blotting analyses of recombinant GDF6 and GDF7. Recombinant proteins were analyzed under reduced or nonreduced conditions following blotting with specific antibodies. B, biochemical properties of purified recombinant human BMP10. BMP10 with an N-termi- nal His6 tag was purified from conditioned media of 293T cells using metal chelate chro- matography. BMP10 was detected by Coomassie Blue staining (lanes 1–3) and immuno- blotting using antibodies against polyhistidine His6 (lanes 4 and 5) under nonreduced or reduced conditions. Some samples were treated with peptide N-glycosidase F (PNGase) to remove N-linked carbohydrate side chains.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Recombinant, Purification, Staining

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 3. Treatment with GDF6, GDF7, and BMP10 stimulated the Smad1/5/8 pathway but not the Smad2/3 pathway in mouse MC3T3 cells. Cells were transiently transfected with the BMP-responsive reporter BRE (A) or the TGF/ac- tivin-responsive CAGA reporter (B). At 48 h after transfection, cells were incubated for 20 h in the absence(Ct)orpresenceofGDF6(10nM),GDF7(10 nM), or BMP10 (3 nM). Cells treated with BMP2 (0.1 nM), BMP7 (3 nM), and TGF (0.1 nM) served as pos- itive controls. C, cells were transfected with the BRE reporter and treated with increasing doses of GDF6, GDF7, or BMP10. The relative luciferase activity was normalized based on the -galacto- sidase activity to correct for variations in transfec- tion efficiency. Results are presented as the mean S.E. D, immunoblotting analysis of Smad1 and Smad2 phosphorylation was performed using cell extracts following treatment of MC3T3 cells with BMP2 (0.1 nM), TGF (0.1 nM), GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). Treatment with GDF6, GDF7, or BMP10 induced the phosphoryla- tion of Smad1 (top) but not Smad2 (Bottom). BMP2 and TGF served as positive controls. Arrows indi- cate the immunoreactive bands of phosphoryl- ated Smad proteins. Ct, control.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Incubation, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 4. Antagonistic effects of inhibitory Smad proteins on the stimulation of the BRE promoter by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of plasmids encoding the inhibitory Smad6 or Smad7. After transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), BMP10 (3 nM), or BMP2 (0.1 nM), before determina- tion of luciferase activity. The relative luciferase activity was normalized based on the -galactosidase activity to correct for variations in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 5. Overexpression of ALK3 or ALK6 confers ligand signaling by GDF6, GDF7, or BMP10. A, COS7 cells were transfected with 500 ng of the BRE reporter construct together with 30 ng of different ALK plasmids or increasing amounts of the ALK3 or ALK6 plasmid. After 5 h of incubation, cells were treated with GDF6 (1 nM), GDF7 (1 nM), or BMP10 (3 nM) for 24 h. The relative luciferase activity was normalized based on the -galactosidase activity. Results are presented as the mean S.E. B, confirmation of the overexpression of different human ALK receptor transcripts in transfected cells using semi-quantitative PCR. , transfection with the empty vector; , transfection with individual ALK plasmid. In select cases, the endogenous monkey transcripts were amplified in COS7 cells because of sequence conservation.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Amplification, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 7. Overexpression of the ALK3 shRNA suppressed the stimulation of BRE promoter activities by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the ALK3 shRNA or control shRNA. Two days after transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). As a negative control, cells were transfected with the ALK3 shRNA before treatment with TGF (0.1 nM). The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, shRNA, Transfection, Plasmid Preparation, Control, Incubation, Negative Control, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 8. Overexpression of BMPRII shRNA or ActRIIA shRNA blocked the stimula- tion of the BRE promoter activity by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the BMPRII, ActRIIA, or control shRNA. As a negative control, cells were transfected with the BMPRII shRNA or ActRIIA shRNA before treatment with TGF. The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, shRNA, Activity Assay, Transfection, Plasmid Preparation, Control, Negative Control