Journal: PLoS ONE

Article Title: Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

doi: 10.1371/journal.pone.0081713

Figure Lengend Snippet: Oligonucleotide primers used in quantitative and non-quantitative PCR.

Article Snippet: Immunoprecipitated proteins were eluted from beads with 200 μl of 0.1 M Na-citrate pH 2.5, neutralized with NaOH, mixed with 50 μl of 5 x Laemmli sample buffer and boiled for 5 min. Eluted protein samples were separated on a 15% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an anti-myostatin antibody (sc-28910, Santa Cruz Biotechnology).

Techniques:

Journal: PLoS ONE

Article Title: Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

doi: 10.1371/journal.pone.0081713

Figure Lengend Snippet: (A) A representative Northern blot identified canonical myostatin (Mstn) and MSV mRNAs in poly(A) + RNA isolated from sheep skeletal muscle using a radiolabeled probe complementary to exon 1 & 2 sequence of sheep myostatin (nt 1–621). (B) Schematic representation of alternative splicing of the myostatin gene. Genomic structure, splicing of canonical myostatin and MSV mRNAs are shown as determined by RT-PCR amplification and sequencing. The sheep myostatin gene has a cryptic third intron sequence (Int 3, 1011 bp) located 21 bp downstream of the intron 2/exon 3 boundary, thereby removing the coding sequence of the canonical mature myostatin protein. Alternate splicing creates a new ORF (966 bp) by appending a novel C-terminal coding sequence (exon 3b, 198 bp) to a truncated propeptide coding sequence of myostatin (exon 1 & 2 and 3a) in the MSV transcript. Open boxes show 5′ and 3′ untranslated regions, filled boxes represent translated sequences. Also shown are exons (Ex), introns (Int), translation start (ATG) and stop (TGA, TAA) sites, and the size of each transcript. Location of the 11 bp deletion in exon 3 identified in Belgian Blue cattle is also indicated. (C) Tissue-specific mRNA expression of MSV and myostatin was analyzed in biceps femoris (Biceps), quadriceps (Quad.) and semitendinosus (Semit.) muscles, and heart, liver, brain, kidney, testicle, ovary, gut, skin and aorta tissues of three months old sheep using RT-PCR. Actin was used as a positive control for each tissue sample. NTC is a no template PCR control. (D) Multiple polypeptide sequence alignment of the predicted C-terminus of MSV in sheep, cattle, pig and dolphin. A consensus proteolytic cleavage site [(K/R)-(X) n -(K/R)↓ where n = 0, 2, 4, 6 and X is any amino acid except cysteine at aa 271–274] has been identified for precursor convertases. A dotted line indicates the location of the putative cleavage site. The scale shows the positions of the amino acid residues in the MSV polypeptide sequence. The unshaded background highlights residues that are different from the consensus sequence. An in-silico predicted secondary structure of mature sheep MSV is also shown. (E) Schematic representation of the known and proposed proteolytic processing of canonical myostatin and MSV precursors, respectively. The location of the secretion signal peptide and the C-terminal cleavage sites are indicated. Grey filling shows the novel C-terminus of the MSV precursor. Black bars denote the location of polypeptide sequences used to raise MSV-specific polyclonal antibodies (MSVab and MSVab65). (F) Detection of MSV-immunoreactive proteins in semitendinosus muscles of sheep and cattle and its absence in gastrocnemius muscles of mouse and rat (20 µg of total protein per lane) using an anti-MSVab in Western immunoblotting. Recombinant peptide (Recomb.) corresponds to a polypeptide for the C-terminal 65 amino acids (11.9 kDa) of sheep MSV. Molecular weights of a protein marker are also indicated.

Article Snippet: Immunoprecipitated proteins were eluted from beads with 200 μl of 0.1 M Na-citrate pH 2.5, neutralized with NaOH, mixed with 50 μl of 5 x Laemmli sample buffer and boiled for 5 min. Eluted protein samples were separated on a 15% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an anti-myostatin antibody (sc-28910, Santa Cruz Biotechnology).

Techniques: Northern Blot, Isolation, Sequencing, Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Muscles, Positive Control, Control, In Silico, Western Blot, Recombinant, Marker

Journal: PLoS ONE

Article Title: Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

doi: 10.1371/journal.pone.0081713

Figure Lengend Snippet: (A) Co-immunoprecipitation (Co-IP) of myostatin protein was performed with an MSV-specific rabbit polyclonal antibody (MSVab) with normal rabbit IgG serving as a control (IgG) from a sheep muscle protein extract (Muscle). MSVab Co-IP was carried out with or without rMSV-V5 protein (rMSV). Protein samples were separated on a 15% SDS- PAGE, transferred to a nitrocellulose membrane and probed with an anti-myostatin antibody. Myostatin protein bands (Mstn) at 13 and 26 kDa are indicated with arrows. (B) Characterization of the interaction of mature MSV with mature myostatin using surface plasmon resonance assay. A representative sensorgram is shown for rMSV (6.25–100 nM) binding to and dissociating from myostatin immobilized on a sensorchip. For each concentration, association and dissociation were measured in duplicate and the response units (RU) are plotted against time. (C) Ratios of phosphorylated (Ser 423/425 ) to total Smad2/3 protein abundance in nuclear protein fractions of proliferating myoblasts of the MSV-line and Control-line treated with or without myostatin (8 nM, n = 3). Unlike letters indicate significance (P<0.05). (D) Ratios of phosphorylated (Ser 423/425 ) to total Smad2/3 protein abundance in cytoplasmic protein fractions of proliferating myoblasts of the MSV-line and Control-line treated with or without myostatin (8 nM, n = 3). (E) CAGA-luciferase reporter assay, wherein the MSV-line and Control-line were treated with or without myostatin (8 nM) for 24 hours (n = 3). Firefly luciferase luminescence values were normalized to renilla luciferase internal control and expressed as fold induction to vehicle controls of the Control-line. (F) Arbitrary concentrations (mean ± S.E.M.) of MSV and myostatin mRNAs in semitendinosus muscle of male sheep at 1, 3, 6, 9, 12 and 18 months of age (n = 6 per age) determined using qPCR. (G) The abundance (mean ± S.E.M.) of MSV (37 kDa) and myostatin (26 kDa) proteins during post-natal muscle growth in semitendinosus muscle of male sheep determined using Western immunoblotting at 1, 3, 6, 9, 12 and 18 months of age (n = 6 per age). α-tubulin (55 kDa) was used to assess the uniformity of loading. (H) A proposed model for the regulation of myoblast proliferation by MSV and myostatin (Mstn).

Article Snippet: Immunoprecipitated proteins were eluted from beads with 200 μl of 0.1 M Na-citrate pH 2.5, neutralized with NaOH, mixed with 50 μl of 5 x Laemmli sample buffer and boiled for 5 min. Eluted protein samples were separated on a 15% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an anti-myostatin antibody (sc-28910, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Control, SDS Page, Membrane, SPR Assay, Binding Assay, Concentration Assay, Quantitative Proteomics, Luciferase, Reporter Assay, Western Blot

Journal: PLoS ONE

Article Title: Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

doi: 10.1371/journal.pone.0081713

Figure Lengend Snippet: Surface plasmon resonance kinetic analysis on the binding of MSV to myostatin, ActRIIB to myostatin and myostatin to itself.

Article Snippet: Immunoprecipitated proteins were eluted from beads with 200 μl of 0.1 M Na-citrate pH 2.5, neutralized with NaOH, mixed with 50 μl of 5 x Laemmli sample buffer and boiled for 5 min. Eluted protein samples were separated on a 15% SDS-PAGE gel, transferred to a nitrocellulose membrane and probed with an anti-myostatin antibody (sc-28910, Santa Cruz Biotechnology).

Techniques: SPR Assay, Binding Assay

Journal: Genes

Article Title: Efficient and Specific Generation of MSTN -Edited Hu Sheep Using C-CRISPR.

doi: 10.3390/genes14061216

Figure Lengend Snippet: Figure 2. Phenotype and molecular analysis of MSTN-edited Hu sheep. (A) Pictures of MSTN-KO and control male sheep. The top and bottom pictures were taken from the lateral view and the rear side view, respectively. Red arrows indicate muscular protrusion, intermuscular boundaries, and grooves in the forequarters and hindquarters of MSTN-KO sheep. (B) Growth curve of MSTN- KO and control Hu sheep from birth to 8 months. Shown are mean values ± SEM. * p < 0.05. (C,D) HE-stained cross section and myofiber size of gluteus muscles from MSTN-KO (#521) and control sheep. **** p < 0.001. (E) Western blot analysis of MSTN, AKT, p-AKT, ERK1/2, p-EKR1/2, p38, p-P38 and GAPDH. The number under the blot picture is the relative protein expression relative to the control group.

Article Snippet: The proteins were subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany) and probed with primary antibodies against GAPDH (60004-1-Ig, Proteintech, Wuhan, China), MSTN (19142-1-AP, Proteintech), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2 (4370T, Cell Signaling Technology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), P38 (9272S, Cell Signaling Technology), and p-P38 (8690S, Cell Signaling Technology).

Techniques: Control, Staining, Muscles, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of a Novel Pool of Extracellular Pro-myostatin in Skeletal Muscle

doi: 10.1074/jbc.m706678200

Figure Lengend Snippet: FIGURE 5. LTBP-3 increases skeletal muscle fiber area and reduces myo- statin-induced signaling. A, LTBP-3 increases skeletal muscle fiber area. Adult mouse tibialis anterior muscles were transfected by electroporation with GFP and LTBP-3 as indicated. Transverse sections of muscles were ana- lyzed for the cross-sectional area of GFP positive fibers. Histogram indicates the distribution of cross-sectional areas of fibers expressing GFP and LTBP-3 (red bars) and GFP alone (green bars). *, Student’s t test p 0.005. Images depict GFP expressing fibers (green), GFP and LTBP-3 expressing fibers (red), and nuclei (blue). B, LTBP-3 reduces myostatin induced phosphorylation of Smad2. Xenopus embryos were injected with LTBP-3 and myostatin RNA, as indicated and kept at 23 °C. Animal caps were cut 3 h after RNA injection and harvested 4 h after dissection. Samples were detected by immunoblot, as indicated.

Article Snippet: Anti-HA-peroxidase rat monoclonal peroxidase-conjugated antibody (clone 3F10; Roche); anti-FLAG-peroxidase mouse monoclonal peroxidase-conjugated antibody (Sigma); antiMyc-peroxidase mouse monoclonal peroxidase-conjugated antibody (Roche); anti-actin mouse monoclonal antibody (A4700, Sigma); anti-P-Smad2 rabbit polyclonal antibody (Cell Signaling Technologies, Danvers, MA); anti-mouse GDF-8 propeptide sheep polyclonal antibody (AF1539, R&D); and anti-mouse GDF-8 goat polyclonal antibody (AF788, R&D).

Techniques: Muscles, Transfection, Electroporation, Expressing, Phospho-proteomics, Injection, Dissection, Western Blot