C2C12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) foetal bovine serum with 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B. To induce differentiation, cells were grown to 85-90% confluence, and the medium was changed to DMEM with 2% horse serum supplemented with penicillin, streptomycin and amphotericin B as described above. FLAG-tagged p38α and p38β were acquired from Addgene (Cambridge, MA, USA) . Constructs were transfected into C2C12 myoblasts with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. To inhibit p38 activity, SB 202190 (Promega, Madison, WI, USA) solubilised in dimethyl sulphoxide (DMSO) or DMSO as control was added to differentiation media at 48 hours following the induction of differentiation, and medium with inhibitor was changed daily.
Cells were fixed with 4% formaldehyde and stained with the following antibodies: Flag M2 (1:1,000 dilution; Sigma-Aldrich, St Louis, MO, USA), myosin heavy chain (MyHC) (1:20 dilution; Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, IA, USA), Alexa Fluor 488 goat anti-mouse antibody (1:1,000 dilution; Molecular Probes/Invitrogen) and Alexa Fluor 555 goat anti-mouse antibody (1:1,000 dilution; Molecular Probes/Invitrogen). The differentiation index was calculated as the number of MyHC-positive nuclei divided by the total number of nuclei. The fusion index was quantified as the number of nuclei per MyHC-positive cell. Five fields of view at ×20 magnification were counted and averaged per replicate, with a total of three replicate experiments.
Statistical analyses were performed using StatPlus software (AnalystSoft Inc; http://www.analystsoft.com/en/products/statplus/). The data shown are means with SD, and Student's t-tests were performed to determine significance for the differentiation and fusion indices.
Western blot analysis
For Western blot analysis, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris∙HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM NaF, 200 μM Na3VO4, 1 mM phenylmethanesulphonylfluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml leupeptin and 10 μg/ml pepstatin), and 5× Laemmli buffer (300 mM Tris, pH 6.8, 0.01% bromophenol blue, 10% SDS, 50% glycerol and 5% β-mercaptoethanol) was added to 25 μg of protein per sample to a final concentration of 1× for SDS-PAGE. For fractionation, cells were lysed in hypotonic buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 50 mM NaF, 200 μM Na3VO4, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 10 μg/ml pepstatin) and left on ice for 10 minutes. Cells were then passed through a 25-gauge needle three times and centrifuged at 500 g to pellet nuclei and unlysed cells. The supernatant was collected as whole cytoplasm. For further fractionation of the cytoplasm, the supernatant was centrifuged again at 5,000 g to pellet mitochondria and membrane fractions. The supernatant was then collected and centrifuged at 100,000 g to pellet any remaining cell particles, and the resulting supernatant was collected as cytosol. RIPA buffer was added to all fractions to a final concentration of 1× for complete lysis. Recombinant phosphorylated p38α (Millipore, Billerica, MA, USA) was dephosphorylated using λ-protein phosphatase (New England Biolabs, Inc, Ipswich, MA, USA) by adding 800 U of phosphatase to 200 ng of p38α diluted in λ-phosphatase buffer, and the sample was assayed at 30°C for 1 hour. Antibodies used for blotting were as follows: α-actinin (1:125 dilution; Abcam, Cambridge, MA, USA), COX IV (1:1,000 dilution; Abcam), GRP78 (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA), lamin A/C (1:500 dilution; Abcam), MyHC (1:100 dilution; Developmental Studies Hybridoma Bank), MyoD (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), myogenin (1:100 dilution; Developmental Studies Hybridoma Bank), neural cell adhesion molecule (1:200 dilution; Abcam), p38α (1:500 dilution; Cell Signaling Technology), phospho-p38 (1:500 dilution; Abcam) and β-tubulin (1:1,000 dilution; Developmental Studies Hybridoma Bank). An Alpha Innotech HD2 imaging system (R&D Systems, Minneapolis, MN, USA) was used to quantify phospho-p38 and tubulin expression.
FSBA treatment and substrate labelling
Cells were lysed in Nonidet P-40 buffer (50 mM Tris∙HCl, pH 7.8, 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 10 μg/ml pepstatin). Lysate was treated at a concentration of 2 mg/ml with 20 mM 5'-4-fluorosulphonylbenzoyladenosine (FSBA) solubilised in DMSO and placed at 30°C for 1 hour. The sample was then diluted down to 1:5 with Nonidet P-40 buffer minus protease inhibitors and desalted using Millipore Amicon ultrafiltration columns with a 10 kDa molecular weight cutoff. Following concentration, the sample was diluted to 4 mg/ml with Nonidet P-40 buffer and diluted 1:2 with 2× kinase assay buffer (40 mM 3-morpholinopropane-1-sulphonic acid (MOPS), pH 7.2, 50 mM β-glycerophosphate, 10 mM ethylene glycol tetraacetic acid (EGTA), 2 mM Na3VO4, 2 mM DTT, 50 mM MgCl2, 400 μM cold ATP and 5 μCi [γ-32P]ATP). Recombinant p38α or p38β (Millipore) was added to a final concentration of 0.5% (wt/wt) total protein. Control and kinase-added samples were assayed at 30°C for 1.5 hours. For one-dimensional SDS-PAGE, 5× Laemmli buffer was added following the assay to 1×, and the sample was electrophoresed. For two-dimensional electrophoresis, 17-cm ReadyStrip immobilised pH gradient (IPG) strips (Bio-Rad Laboratories, Hercules, CA, USA) were directly rehydrated with labelled lysate diluted in rehydration buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) and 1% DTT) following the manufacturer's directions. Isoelectric focusing was performed on a PROTEAN IEF Cell (Bio-Rad Laboratories) under the following conditions: 200 V for 1 hour, 500 V for 1 hour, 5,000 V ramp for 5 hours and 5,000 V for 80,000 VH. IPG strips were then equilibrated following the manufacturer's instructions and overlaid onto a 12% SDS-PAGE gel. Following electrophoresis, gels were dried and imaged. For one-dimensional electrophoresis, 100 μg of lysate was used per reaction. For two-dimensional electrophoresis, 300 μg of lysate was used.
For in vitro substrate identification, assays were performed as described above with the following modifications. A quantity of 1.5 mg of lysate was treated with FSBA, the sample was desalted and 2× kinase assay buffer was added (40 mM MOPS, pH 7.2, 50 mM β-glycerophosphate, 10 mM EGTA, 2 mM Na3VO4, 2 mM DTT, 50 mM MgCl2 and 2 mM cold ATP). The sample was then split into three 500-μg aliquots, and 5 μg of heat-inactivated p38α was added to the control, 5 μg of active p38α was added to the second aliquot and 5 μg of active p38β was added to the third aliquot. The samples were then assayed for 3 hours at 30°C.
For in vivo substrate identification, assays were performed as above with the following modifications. At 48 hours of differentiation, cells were treated with 5 μM of SB 202190 or an equivalent amount of DMSO as vehicle. Twenty-four hours later the cells were lysed in Nonidet P-40 buffer. Lysate (1 mg) from DMSO-treated cells or 2× 1 mg of lysate from SB 202190-treated cells was inhibited with FSBA, the samples were desalted and 2× kinase assay buffer was added to each. A quantity of 5 μg of active p38α was added to one of the lysate aliquots from SB-treated cells. The samples were then assayed for 3 hours at 30°C.
After assaying the samples, they were precipitated by methanol chloroform, then redissolved in 200 μl of 8 M urea and 50 mM Tris∙HCl, pH 8.1, with sonication. The samples were then reduced with 20 mM DTT for 1 hour at 60°C and alkylated by 100 mM iodoacetamide for 30 minutes at room temperature in the dark. Subsequently, the samples were diluted to 2 M urea with 50 mM Tris∙HCl, pH 8.1, and digested with trypsin at a protein-to-trypsin ratio of 50:1 (wt/wt) for 16 hours at 37°C. Next, the digested samples were acidified to pH 2 using 10% (vol/vol) formic acid. Dimethyl labelling of the samples was performed as reported previously  and is described briefly as follows. The acidified peptides were loaded onto C18 solid phase extraction (SPE) columns (50 mg of packing material). After brief washing with 50 mM sodium phosphate buffer, pH 7.5, 3 ml of light, intermediate and heavy labelling reagents were loaded onto C18 SPE columns trapped with control, p38α- and p38β-labelled samples, respectively. After being washed with 0.1% (vol/vol) formic acid, the labelled samples were eluted with 80% acetonitrile (ACN) (vol/vol) and 0.1% (vol/vol) formic acid, then dried by vacuum centrifugation.
Phosphopeptide enrichment by TiO2 was carried out as reported previously  with modifications. The dried samples were redissolved with 65% ACN/2% trifluoroacetic acid (TFA)/saturated glutamic acid and combined. TiO2 beads suspended in 65% ACN/2% TFA/saturated glutamic acid were added into the above samples with a peptide to TiO2 bead ratio of 1:4 (wt/wt). After being nutated for 40 minutes, the TiO2 beads were recovered by centrifugation and washed thoroughly with 65% ACN/2% TFA. Finally, the enriched phosphopeptides were eluted with 10% (vol/vol) NH3·H2O and dried by vacuum centrifugation.
Online liquid chromatography tandem mass spectrometry analysis
Online liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed as reported previously [13, 14] with modifications. The dried sample was redissolved with 0.1% formic acid and loaded onto a biphasic trap column (200 μm ID × 10 cm; 5-cm reversed phase column packed with ReproSil-Pur C18 resin (5 μm at 200 Å; Dr.Maisch GmbH, Ammerbuch-Entringen, Germany) and a 5-cm monolith strong cation exchange (SCX) column). The trapped phosphopeptides were eluted from the trap column onto a C18 tip column (75 μm ID × 20 cm, 3 μm at 200 Å; Dr.Maisch GmbH) by a series of salt washes at increasing concentrations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200 and 1,000 mM). Each fraction was then separated by reversed phase-based gradient elution and detected using an LTQ Orbitrap XL Fourier transform mass spectrometer (Thermo Scientific, Waltham, MA, USA). The reversed phase gradient was set as follows: 0% to 5% ACN for 2 minutes, 5% to 30% ACN for 90 minutes and 30% to 80% ACN for 5 minutes. After flushing with 80% ACN for 10 minutes, the column was equilibrated with 0.1% formic acid aqueous solution for 13 minutes. The LTQ Orbitrap XL Fourier transform mass spectrometer was operated in positive ionization mode. A voltage of 1.8 kV was applied. MS and MS/MS spectra were acquired in a data-dependent mode, and one full MS scan was followed by ten MS/MS scans. The resolution was set at 60,000 at m/z 400 after accumulation to a target value of 500,000.
Protein identification and quantification
All MS/MS spectra in one acquired raw file were converted to a single *.mgf file using DTASuperCharge version 2.0a7 (Matrix Science, Boston, MA, USA). The *.mgf file was queried against the International Protein Index mouse database version 3.52 (EMBL-EBI; http://www.ebi.ac.uk/IPI/IPIhelp.html) using Mascot version 2.1 (Matrix Science). To evaluate the false discovery rate (FDR), reversed sequences were appended to the database. Cysteine residues were searched as a static modification of +57.0215 Da; methionine residues were searched with a variable modification of +15.9949 Da; and serine, threonine and tyrosine residues were searched with a variable modification of +79.9663 Da. Light, intermediate and heavy dimethylation of peptide amino termini and lysine residues were set as variable modifications of +28.0313 Da, +32.0564 Da and +36.0757 Da, respectively. Peptides were queried using full tryptic cleavage constraints with up to two missed cleavage sites. The mass tolerances were 7 ppm for parent masses and 0.5 Da for fragment masses. Phosphopeptides with a Mascot score ≥ 30 (rank 1, P ≤ 0.05, bold red required) were selected and quantified (FDR < 0.01). Phosphorylation site localisation and phosphopeptide quantification were performed using a dimethyl-adapted version of MSQuant version 2.0a81. For each peptide, the putative site of phosphorylation yielding the highest posttranslational modification (PTM) score was accepted (PTM score > 13 required, as described previously ). Peptide ratios were obtained by calculating the extracted ion chromatograms of the light, medium and heavy forms of the peptide using the monoisotopic peaks only, and protein ratios were calculated from the average of all quantified peptides. All MSQuant outputs of the same online multidimensional separation were then imported into StatQuant version 1.2.2, and the quantified phosphopeptides were sorted together and exported.
1 μg recombinant HSP27 (Enzo Life Sciences, Farmingdale, NY, USA), recombinant Rps27 (Abnova, Taipei City, Taiwan) or recombinant Smad9 (Abnova) were diluted in 30 μl of 1× kinase assay buffer (20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM DTT, 25 mM MgCl2, 200 μM cold ATP and 2.5 μCi [γ-32P]ATP). A quantity of 500 ng of p38α or p38β was then added, and the samples were assayed at 30°C for 1 hour. Laemmli buffer at 5× concentration was added to 1× to terminate the assays, then the samples were electrophoresed and the gels were dried and imaged. Peptide assays were performed similarly with 5 μg of the following peptides synthesised by Biomatik (Cambridge, ON, Canada): HSP27 S180-APLPKAVTQSAEITIPVTF, HSP27 A180-APLPKAVTQAAEITIPVTF, Rps27 S27-KHKKKRLVQSPNSYFMDVK, Rps27 A27-KHKKKRLVQAPNSYFMDVK, Smad9 T136-NPYHYQRVETPVLPPVLVP and Smad9 A136-NPYHYQRVEAPVLPPVLVP. The frequency logo was generated using WebLogo .