Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc associates with GDF11 and MSTN. Protein-protein interactions between GDF11PRO-Fc or MPRO-Fc and rGDF11, rMSTN, or rActivin A were determined by a pull-down assay. GDF11PRO-Fc or MPRO-Fc was incubated with rGDF11, rMSTN, or rActivin A for 1 h at 4 °C. Fc-fused protein complexes were separated on a protein A/G-coated agarose resin and eluates were run on a 12% SDS-PAGE gel under reducing conditions and probed by western blot. Input control was 5% of the input material. WB western blot

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Protein-Protein interactions, Pull Down Assay, Incubation, SDS Page, Western Blot, Control

Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc blocks GDF11/MSTN-induced myotube atrophy in C2C12 cells. a Schematic detailing experimental timeline in C2C12 myotubes. AAV6-EGFP or AAV6-GDF11PRO-Fc was added to C2C12 myotubes at a MOI of 10 on day 5 post-differentiation and 100 ng/ml rGDF11 or rMSTN was added on day 7. Myotubes were stained and analyzed on day 10. b EGFP expression was evident at 48–72 h in C2C12 myotubes treated with AAV6-EGFP (MOI 10 ). Scale bars represents 50 μm. c Vector genome copy number per diploid genome in C2C12 myotubes 72 h after addition of AAV6-EGFP or AAV6-GDF11PRO-Fc (MOI 10 ). d Representative immunofluorescence images of C2C12 myotubes. C2C12 myotube membranes were visualized by staining with an anti-dystrophin antibody (red). Nuclei were stained with DAPI (blue). Inset shows a zoomed-in region. Scale bars represent 50 μm (main panel) and 25 μm (panel inset). e The fraction of nuclei incorporated into myotubes (differentiation index) was calculated and presented as a percentage of control. f Average myotube diameter relative to control and ( g ) distribution of diameter measurements. For myotube diameter measurements, each myotube was measured at three points along the length of the myotube and averaged. h Number of nuclei incorporated per myotube. A minimum of 50 myotubes were analyzed per experimental condition. i pSMAD2/3 relative to tSMAD2/3 was assessed by western blot. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. Data represents results from three separate experiments. All error bars represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant; compared to AAV6-EGFP-treated control. † p < 0.05; †† p < 0.01; ††† p < 0.001; compared to AAV6-EGFP + ligand-treated. pSMAD2/3: phosphorylated SMAD2/3; tSMAD2/3: total SMAD2/3

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Staining, Expressing, Plasmid Preparation, Immunofluorescence, Control, Western Blot