Journal: Biochemical and Biophysical Research Communications

Article Title: T3 affects expression of collagen I and collagen cross-linking in bone cell cultures

doi: 10.1016/j.bbrc.2010.08.022

Figure Lengend Snippet: T3 stimulated mRNA expression of genes related to collagen cross-linking process. Cells were cultured in absence (Co) or presence of 10 −7 M T3 for 4, 8 and 12 days. Compared to untreated controls T3 increased expression of Lox (A), Plod2 (B) and Bmp1 (C) significantly. Total RNA was isolated and subjected to QRT-PCR, which was performed as triplicate. The bars indicate mean ± SD of a typical experiment. ∗ P ⩽ 0.05; ∗∗∗ P ⩽ 0.001 ( n = 3).

Article Snippet: The obtained cDNA was subjected to PCR amplification with a real time cycler using TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan primers (Applied Biosystems) that amplify lysyloxidase (Lox, Mm00495386_m1) bone morphogenetic protein 1 (Bmp1, Mm00802225_m1) and procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2, Mm00478767_m1).

Techniques: Expressing, Cell Culture, Isolation, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

doi: 10.1371/journal.pgen.1006019

Figure Lengend Snippet: (A) Muscle lysates were fractionated from total lysate (T) into cytosolic (C), light microsome (L) and heavy microsome (H) components. Individual fractions were immunoblotted with an antibody against the active domain of myostatin. There was less myostatin expressed in the soluble fractions of TG+ mdx mice muscle lysates compared to mdx controls. (B) A phylogenetic tree of mouse TGFβ family members derived from protein alignments illustrates the high similarity between these proteins, and explain why antibodies to myostatin cross react to GDF11. (C, D) Mouse LTBP4 and either myc-epitope-tagged myostatin (C) or myc-tagged GDF11 (D) were heterologous expressed in HEK293T cells. The myc-tagged myostatin and GDF11 were the complete prodomains and active domains together. Co-immunoprecipitation was performed on cell lysates by immunoprecipitating with anti-LTBP4 antibody followed by immunoblotting with anti-myc antibody detecting myostatin or GDF11. A co-IP control for each experiment was performed without adding IP antibody (labeled as “No anti”). LTBP4 was found to associate with both myostatin and GDF11 in vitro. The 49 KDa band in the first left lane of panels C and D likely represents endogenous myc.

Article Snippet: Mouse cDNA clones of Mstn (NM_010834), Gdf11 (NM_010272), Tgfβ1 (NM_011577), Tgfβ2 (NM_009367), and Tgfβ3 (NM_009368) were purchased from Origene (catalog numbers MR227629, MR223819, MR227339, MR225633, and MR206441, respectively).

Techniques: Derivative Assay, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Control, Labeling, In Vitro

Journal: PLoS Genetics

Article Title: Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

doi: 10.1371/journal.pgen.1006019

Figure Lengend Snippet: RNA sequencing reveals relative abundance of TGFβ family members in muscle.

Article Snippet: Mouse cDNA clones of Mstn (NM_010834), Gdf11 (NM_010272), Tgfβ1 (NM_011577), Tgfβ2 (NM_009367), and Tgfβ3 (NM_009368) were purchased from Origene (catalog numbers MR227629, MR223819, MR227339, MR225633, and MR206441, respectively).

Techniques: RNA Sequencing

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 1. cFN and pFN bind BMP1. A, 80 ng of FLAG-tagged BMP1 was preincubated overnight with an equimolar amount of cFN or pFN. The com- plex was then immunoprecipitated (IP) with antisera raised against the N-ter- minal 70-kDa FN fragment (anti-70K), which recognizes both cFN and pFN. Immunoblotting (IB) was performed using either monoclonal anti-FLAG or polyclonal anti-70K antibody. B, BMP1 binding of cFN and pFN in ELISA. BMP1 at 3.4, 11.4, 34.1, 113.6, 340.6, and 1136.4 nM was incubated with cFN or pFN adsorbed to microtiter wells, as described under “Experimental Procedures.” The calculated KD values for BMP1 binding to cFN and pFN, as determined by Scatchard-typeplots,were11020and12030nM,respectively.Valuesare expressed as the mean S.D. of three experiments.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 3. FN binds BMP1 at multiple sites. FLAG-tagged BMP1 was preincubated overnight with various truncated forms of FN followed by immunoprecipitation (IP). A, immunoblots (IB) show that immunopre- cipitation of either the FN N-terminal 70-kDa fragment (70K) or the FN C-terminal fragment (III1-C) using anti-70K antibody or monoclonal LabMab anti-FN antibody, respectively, co-precipitated BMP1. B, immu- noblots show that immunoprecipitation of III1-C(EDA) or III1-C(EDA), with monoclonal LabMab anti- body, co-precipitated similar amounts of BMP1. C, immunoblots show that immunoprecipitation of III1–14 with monoclonal LabMab antibody co-precipitated BMP1. D, a schematic is shown of the protein domain structure of FN and of truncated forms of FN used in this study. FN types I, II, and III repeats are shown. Alternatively spliced regions EDA, EDB, and V sequences are indicated, as are linker sequences (horizontal lines).

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 2. BMP1 binds FN via its non-protease domain sequences. A, an immunoblot (IB) shows input amounts of cFN, FLAG-tagged versions of full-length BMP1, a form of BMP1 containing all domains except the protease domain (BMP1-D), and isolated BMP1 protease domain (BMP1-P). B, after overnight incuba- tion of BMP1-D or BMP1-P with cFN, an immunoblot shows that the immunoprecipitation (IP) of cFN using anti-70K antibody pulls down full-length BMP1 and BMP1-D but not BMP1-P.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Western Blot, Isolation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 4. FN enhances BMP1 proteolytic processing of probiglycan, pro- collagen type I, and Chordin in vitro. Thirty-minute in vitro cleavage assays of probiglycan (A) or 6-h cleavage assays of type I procollagen (B and C) or Chordin (D) were performed in cFN-coated wells, with BSA-coated wells used as controls (A–C) or in the presence or absence of soluble cFN (D). A, probig- lycan (ProBgn) and mature biglycan (Bgn) were detected by immunoblot (IB) using antibody (LF51) directed against sequences in the mature region of biglycan (20). B and C, procollagen, processing intermediates, and mature 1(I) chains were detected by immunoblot using C-telopeptide polyclonal antibody LF67 (20). Samples in B and C were run on 7.5 and 6.0% acrylamide SDS-PAGEgels,respectively,thelattertoseparatepC-1(I)andpN-1(I)proc- essing intermediates. In B, in which reactions went somewhat further than in C, mature 1(I) chains are clearly discernable. D, BMP1 processing of C-termi- nal FLAG-tagged Chordin was monitored by immunoblot using anti-FLAG antibody. BMP1 cleaves Chordin at both N- and C-terminal sites (18). Bands for full-length Chordin (chd) and for a processing intermediate cleaved at the N-terminal but not the C-terminal site (C-chd) are marked but were not well separated in the 15% gel. Upon cleavage by BMP1, both bands disappeared, with the appearance of the 15-kDa Chordin C-terminal cleavage product (denoted by an asterisk), indicating complete Chordin processing.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: In Vitro, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 5. Immunofluorescent co-localization of BMP1 and FN in fibro- blast ECM. Paraformaldehyde-fixed cultures of human neonatal fibroblasts, either permeabilized or not permeabilized with Triton X-100, were immuno- stained with antibodies to FN (green) and BMP1 (red) and were stained with 4,6-diamidino-2-phenylindole (DAPI, blue) for localization of nuclear DNA. The yellow signal indicates areas in which green and red signals overlap and, thus, areas of co-localization between FN and BMP1.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Staining

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 6. Detection of endogenous BMP1-FN complexes in cell culture. Conditioned medium from cultures of MG-63 cells pretreated 24 h with TGF1 was subjected to immunoprecipitation (IP) with anti-BMP1 antibody (lane labeled IP BMP1 in panel A), anti-FN antibody (lane labeled IP FN in panelB),orIgG(IPIgG-labeledlanesinbothpanelsAandB)followedbyimmu- noblotting(IB)withanti-FNoranti-BMP1antibody.Inputlanesrepresent30l conditioned media not subjected to immunoprecipitation.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Cell Culture, Immunoprecipitation, Labeling

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 7. Reduced processing of endogenous BMP1 substrates in cul- turesofFN/MEFs.A,confluentFN/andFN/MEFswereincubatedfor 24 h in serum-free medium, and endogenous Chordin (chd) from the condi- tioned medium, concentrated by ethanol precipitation or with heparin- Sepharose, was probed via immunoblotting (IB) using anti-N-terminal-Chor- din antibodies. Staining of ethanol-precipitated media samples with antibodies to secreted proteins sFRP2 and BMP1 shows similar levels of these two secreted proteins in FN/ and FN/ MEF media, arguing against over- all differences in levels of secreted proteins despite different levels of detected Chordin. Staining of cell extracts with anti--tubulin antibody dem- onstrates that FN/ and FN/ MEF cultures contained similar numbers of cells despite different levels of detected Chordin from conditioned media. B, confluent FN/ and FN/ MEFs were treated for 24 h with 50 g/ml ascor- bate and then incubated for 24 h in serum-free medium containing 50 g/ml ascorbatefollowedbyharvestingofmediaandcelllayersamples,whichwere then assayed via Western blot using LF67 antibody directed against the C-te- lopeptide of the 1(I) collagen chain. Positions are shown for procollagen, pN1(I) chains ((pN), processing intermediates from which C-propeptides have been removed but which retain N-propeptides), pC1(I) chains ((pC), processing intermediates from which N-propeptides have been removed but which retain C-propeptides), and mature, fully processed, 1(I) collagen chains. Reduced processing of the C-propeptides of type I procollagen and decreased deposition of mature collagen were observed in FN/ MEF cul- turescomparedwithculturesofFN/MEFs,indicatingdramaticallyreduced pCP activity for BMP1-like proteinases. C, confluent FN/ and FN/ MEFs were incubated for 24 h in serum-free medium, and probiglycan processing was examined in conditioned medium by Western blot using antibody LF51 directed against sequences within mature biglycan (20). Pro and mature forms of biglycan are denoted in the figure as ProBgn and Bgn, respectively. Probiglycan is less processed in FN/ MEF cultures than in FN/ MEF cultures.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Ethanol Precipitation, Western Blot, Staining, Incubation, Activity Assay