Supplemental materials and methods. The itraq labelling of the proteins was done as described previously [1]. Briefly, C2C12 myotubes

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1 1 Supplemental materials and methods 2 itraq labelling and nanolc-ms/ms analysis The itraq labelling of the proteins was done as described previously [1]. Briefly, C2C12 myotubes were treated with Myostatin (5 μg/ml) (or carrier) for 24 hr and washed three times with cold PBS. Two independent itraq experiments were performed. In each itraq experiment, three independent cell cultures were pooled. The myotubes were re-suspended in 200 µl of lysis buffer (500 mm triethylammonium bicarbonate (TEAB), 1% SDS, complete protease inhibitor cocktail tablet (Roche, USA) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche, USA)). The samples were then sonicated using a VCX500 sonicator (Sonics, USA) at 25% amplitude and 8 sec pulse in 3 cycles. After centrifugation at 20,000g for 15 min at 4 o C, proteins in the supernatant were quantified using the BCA Protein Assay kit. Two hundred micrograms of protein from Myostatin treated and control myotubes were reduced by tris (carboxyethylphosphine) hydrochloride (TCEP), alkylated with methylmethanethiosulfonate (MMTS). The samples were then diluted to 0.1% SDS and digested with trypsin as previously reported [2, 3]. The resulting peptides were dried and resuspended in 30 μl of 500 mm TEAB (ph 8.5). Each sample was divided into two equal portions, and then isotopically labelled according to the manufacturer s protocol (Applied Biosystems, USA). The labelling scheme was 114: Myostatin treated; 115: control; 116: Myostatin Treated; 117: Control. The labelled samples were combined, dried and stored at -80 C until further analysis The dried itraq labelled sample were re-suspended in 200 µl SCX buffer A (10 mm KH 2 PO 4 in 25% acetonitrile, ph 3). SCX chromatography was performed on a Shimadzu Prominence TM UFLC unit (Kyoto, Japan) using a mm (5 µm particle sizes, 200 Å pore size) PolySULFOETHYL A TM column (PolyLC, USA). The sample was fractionated using a 50-min gradient of 100% buffer A for 5 min, 0 30% buffer B (10 mm KH 2 PO 4, ph 3.0, 500 mm KCl, ACN/H 2 O 25/75 (v/v)) for 35 min, % buffer B for 5 min and finally 100% buffer B for 5 min, at a constant flow rate of 1 ml/min. Fractions were collected at 1 min intervals and consecutive fractions with low peak intensity were combined. Finally, a total of 20 fractions were obtained, desalted using Sep-Pak C18 cartridges

2 27 28 (Waters Corporation, USA). Eluents were lyophilized and stored at -80 C before LC-MS/MS analysis Each of the dried SCX fractions was reconstituted in 0.1% formic acid and then analyzed with a QStar Elite Q-TOF (Applied Biosystems, Framingham, MA, USA) coupled with a Shimadzu Prominence TM UFLC unit. Each fraction was first trapped and desalted with Zorbax 300SB-C18 column, and further separated with an integrated nano-bored C18 column, packed with Magic 5 μm C18 particles. The UFLC flow rate was set at 30 ml/min and a splitter was used to create an approximate 300 nl/min flow rate at the electrospray tip with a 90-min LC gradient. The data acquisition was performed with Analyst QS 2.0 software (Applied Biosystems, Framingham, MA, USA). The mass spectrometer was set to perform data acquisition with a selected mass range of m/z. The three most abundant peptides (with 2+ to 5+ charge states) were selected for MS/MS analysis, and dynamically excluded for 30 sec with ±30mmu tolerance. Smart information-dependent acquisition (IDA) was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 sec. 41 Data analysis The two independent itraq data sets and the combination of these two data were searched with ProteinPilot software v2.0.1 (Applied Biosystems/MDS-Sciex) for protein identification and quantification. The Paragon algorithm in the ProteinPilot software was used for the peptide identification and further processed by Pro Group algorithm where isoform-specific quantification was adopted to trace the differences between expressions of various isoforms. The defined parameters were as follows: (i) Sample Type: itraq 4-plex (Peptide Labeled); (ii) Cysteine alkylation: MMTS; (iii) Digestion: Trypsin; (iv) Instrument: QSTAR ESI; (v) Special factors: None; (vi) Species: None; (vii) Specify Processing: Quantitate; (viii) ID Focus: biological modifications, amino acid substitutions; (ix) Database: a concatenated target (International Protein Index (IPI) mice database, version 3.34, including sequences and residues) and decoy database [the reverse amino acid sequence for the estimation of false discovery rate

3 (FDR=(decoy_hits/total_hit)*100%]); (x) Search effort: thorough. The peptide for quantification was automatically selected by Pro Group algorithm to calculate the reporter peak area, error factor (EF) and p-value. The p-value can be used to assess whether the changes in protein expression is statistically significant or not. The p-value is a measure of the certainty of a change in the protein expression that is independent of the magnitude of the change. The resulting data set was auto bias corrected and background corrected by the Paragon algorithm. The itraq results of the combined data and the two independent experimental data were exported from ProteinPilot as Protein Summary reports in Microsoft Excel file format non-redundant proteins were identified with FDR lower than 1.0%. Of which, 1290 proteins were identified with Unused ProtScore > 1.3 (Confidence > 95%), and 1337 proteins were quantified with itraq reporter ratio. The itraq result was further analyzed using the online gene ontology software available at and Subcellular and functional categories are based on the annotations of Gene Ontology (GO) using the MGI GO-Slim Chart Tool available at MGI To measure changes in the relative abundance of protein, duplicate sets, [Set #1, 114:115 (Myostatin Treated: Control) and Set #2 116:117 (Myostatin Treated: Control)] were analysed using itraq. This experiment was repeated twice. To assess the reproducibility of the experiments and the quality of the itraq data sets, the geometric mean of itraq reporter ratio and the log scale standard deviation of each identified protein from the four sets of itraq ratio of the two independent experiments were calculated

4 79 References Gan CS, Guo T, Zhang H, Lim SK, Sze SK. A comparative study of electrostatic repulsionhydrophilic interaction chromatography (ERLIC) versus SCX-IMAC-based methods for phosphopeptide isolation/enrichment. J Proteome Res 2008; 7 (11): Datta A, Park JE, Li X et al. Phenotyping of an in vitro model of ischemic penumbra by itraq-based shotgun quantitative proteomics. Journal of proteome research 2010; 9 (1): Guo T, Gan CS, Zhang H et al. Hybridization of pulsed-q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for itraq quantitation. Journal of proteome research 2008; 7 (11):

5 Supplemental figure legends and tables Fig Specific knockdown of MuRF1 expression doesn t alter the expression of sarcomeric genes (Supporting to Fig. 4) RT-qPCR analysis of the expression of individual isoforms of Myh and Myl from untreated and Myostatin treated (Mstn) myotubes from shatrogin-1 (A) or shmurf1 (B) transfected C2C12 cells. Data is from two independent experiments and graphs represent mean fold change when compared to shrna controls. The gene expression changes presented in graphs were found to be insignificant.

6 Table-1 List of primary and secondary antibodies used in this manuscript. Antibodies used in this manuscript Antibodies name Catalog No. Company Atrogin-1 Gift Regeneron MuRF1 Gift Regeneron Myh (all type) MF20 DSHB Myh fast A4.74 DSHB Myh embroynic F1.652 DSHB Myh fast 2A 2F7 DSHB Myh fast 2B 10F5 DSHB Myh fast 2X 6H1 DSHB Myh slow A4.840 DSHB Myl (all type) T14 DSHB IgG agarose A2909 Sigma CBP sepharose GE health α-tubulin T9026 Sigma Ubiquitin sc-8017 Santa Cruz FoxO1 sc Santa Cruz p-foxo1 sc Santa Cruz Smad2/3 sc-6032 Santa Cruz p-smad2/3 sc Santa Cruz Rabbit HRP conjugate Bio-rad Mouse HRP conjugate Bio-rad

7 Table-2 Set of oligo s used for making the shrna/psilencer vector constructs. shrna oligo sequences shrna oligo sequences used for cloning into the psilencer vector shrna Target Sense strand Oligo (5'-3' orientation) Anti-Sense strand Atrogin-1 Set-1 MuRF1 Set-1 Smad3 Set-1 Atrogin-1 Set-2 MuRF1 Set-2 Smad3 Set-2 GATCCGCGCTTCTTGGATGA GAAATTCAAGAGATTTCTCA TCCAAGAAGCGCTT A GATCCGACAAAGAGGATCC GAGTGTTCAAGAGACACTC GGATCCTCTTTGTCTTA GATCCACTTTCTACTGCCAC TTGGTTCAAGAGACCAAGT GGCAGTAGAAAGTTTA GATCCGGAGGTATACAGTA AGGAGTTCAAGAGACTCCTT ACTGTATACCTCCTTA GATCCAGAGGATCCGAGTG GGTTTCTCAAGAGAAAACC CACTCGGATCCTCTTTA GATCCGTTCTCCAGAGTTAA AAGCTTCAAGAGAGCTTTTA ACTCTGGAGAACTT A AGCTTAAGCGCTTCTTGGAT GAGAAATCTCTTGAATTTCT CATCCAAGAAGCGC G AGCTTAAGACAAAGAGGATC CGAGTGTCTCTTGAACACTC GGATCCTCTTTGTCG AGCTTAAACTTTCTACTGCC ACTTGGTCTCTTGAACCAAG TGGCAGTAGAAAGTG AGCTTAAGGAGGTATACAGT AAGGAGTCTCTTGAACTCCT TACTGTATACCTCCG AGCTTAAAGAGGATCCGAGT GGGTTTTCTCTTGAGAAACC CACTCGGATCCTCT G AGCTTAAGTTCTCCAGAGTT AAAAGCTCTCTTGAAGCTTT TAACTCTGGAGAACG

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