Cross-Linking Experiments and MALDI-TOF MS. Acetylcholine receptor (AChR)-rich membranes were purified from Torpedo electric tissue according to Saitoh and Changeux (1). Cross-linking experiments were performed according to Burden et al. (2) by using succinimidyl 4(p-maleimidophenyl)-butyrate (SMPB) that contains N-ethylmaleimide and N-hydroxysuccinimide as reactive groups, which reacted with free sufhydryle and primary amines, respectively. In brief, AChRs-rich membranes were washed with 10 mM sodium phosphate buffer/1 mM EDTA/1 mM EGTA/0.3 mM PMSF/0.02% sodium azide (pH 7.4), pelleted by centrifugation, and resuspended in 10 mM sodium phosphate buffer/1 mM EDTA (pH 8.0) at a final concentration of 4 mg proteins/ml. SMPB in DMSO (2% vol/vol) was added to the membranes at concentrations ranging from 10^–6 to 10^–4 M and incubated in the dark for 30 min at room temperature. Membranes were then pelleted and wash in 10 mM sodium phosphate buffer/1 mM EDTA (pH 8.0) before solubilization in SDS/PAGE sample buffer.

The MALDI-TOF MS analysis of muscle-specific kinase (MuSK) crosslink products was performed after Triton X-100 extraction and immunopurification with anti-cyt MuSK antibody as described in ref. 3. The 125-kDa crosslink product was cut off the gel and treated with trypsin (EC 3.421.4, Roche Diagnostics) as described by Shevchenko et al., (4). Digests were resuspended in 20-m l 1% formic acid, desalted with Zip Tips C18 (Millipore), and eluted with 50% and 80% acetonitrile. The desalted peptide mixture was dried and dissolved in 3-m l 1% formic acid. The matrix used was a saturated solution of 2,5-dihydroxybenzoic acid in 0.1% trifluoroacetic acid. The sample and the matrix (1:1, vol/vol) were loaded on the target by using the dried droplet method. MALDI-TOF spectra of the peptides were obtained with a Voyager-DE STR Biospectrometry Workstation mass spectrometer (PE Biosystems, Framingham, MA). The analyses were performed in positive-ion reflector mode with an accelerating voltage of 20 kV and a delayed extraction of 200 nsec; » 250 scans were averaged. For subsequent data processing, the [scap]data explorer[r] software (PE Biosystems) was used. Spectra obtained for the whole protein were calibrated externaly by using the [M+H]⁺ ion from Des-Arg bradykinin peptide [molecular weight (MW) 904.47] and corticotropin (ACTH) 18–39 fragment peptide (MW 2465.20). The trypsin autoproteolysis product (132–142 fragment [MW 1153.57] and 56–75 fragment [MW 2163.06] ) were used as second calibers. Data mining was performed by using the [scap]profound[r] (The Dialogue Corporation) and [scap]ms-fit[r] (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) programs. A mass deviation of 100 ppm was usually allowed in the database searches. From the 125-kDa crosslink product, four peptides (measured masses: 1036.452, 927.462, 2220.032, and 1188.602) matched with rat 14-3-3 g isoform (residues 79–86, IEMVRAYR; 121–127, VFYLKMK; 143–162, RATVVESSEKAYSEAHEISK; and 218–227, DSTLIMQLLR, respectively). These peptides represent 45 of 247 residues (18%).

Immunofluorescence Microscopy. For indirect immunofluorescence experiments, 3% paraformaldehyde fixed tissues or cells were incubated with primary antibodies and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) as described in ref. 3. In cryostat section of sternomastoide muscle, FITC-conjugated a -bungarotoxin (1 m g/ml; Molecular Probes) was added with the secondary antibody to label the AChRs in the postsynaptic membrane. Samples were mounted in Citifluor (UKC Chemical Laboratory, Canterbury, U.K.). Micrographs were taken with a Leica DMR microscope equipped with a MicroMAX cooled charge-coupled device camera (Princeton Instruments, Trenton, NJ). For double fluorescence pictures, controls confirm that no bleedthrough was detectable under the conditions used (filters L5 for fluorescein, filters TX for Cy3). Digital images were captured by using Meta View Imaging System (Universal Imaging, Downingtown, PA) and arranged using <size;7q>PHOTOSHOP 5.0<size;9q> (Adobe Systems, San Jose, CA). Quantitative analysis of data presented in Fig. 7 was obtained by using Meta View Imaging System. AChR clusters whose longer axis was equal to or greater than 1 m m were scored.

Real-Time Quantitative RT-PCR. The sequences of the primers used for quantitative RT-PCR were as follows: AChR-a , forward 5'-ACCTGGACCTATGACGGCTCt-3' and reverse 5'-AGTTACTCAGGTCGGGCTGGT-3'; AChR-e , forward 5'-CTTGGTGCTGCTCGCTTACTT-3' and reverse 5'-CGTTGATAGAGACCGTGCATT-3'; Utrophin A, forward 5'-GGCAGGAAGATTGCACAAGT-3' and reverse 5'-CTGCTAGCCAAGTCCCAGAG-3'; MuSK, forward 5'-TGAAGCTGGAAGTGGAGGTTTT-3' and reverse 5'-GCAGTAGGGTTACAAAGGAA-3'; Rapsyn, forward 5'-GCAGTGCCATGGAGTGTTGT-3', and reverse 5'-GGCAAAGCAGAGCAGACAGAGT-3'; MCK, forward 5'-GACGAAGGCGAGTGAGAATC-3' and reverse 5'-CATGGAGAAGGGAGGCAATA-3'; ErbB3, forward 5'-CTTACGGGACACAATGCTGA-3' and reverse 5'-GCATGGCTGGAGTTGGTATT-3'; and GAPDH, forward 5'-CAGCAATGCATCCTGCAC-3' and reverse 5'-GCCAAGGTCATCCATGAC-3'.

Assay of Agrin-Induced AChR Clustering. Transfected or untransfected C2C12 cells were cultured on Matrigel-coated (Collaborative Biomedical products) glass coverslips according to the manufacturer’s instructions. Three- to four-day-old myotubes were incubated with agrin [10 ng/ml Cibacron Blue pool (5) for 16 h at 37°C.] To identify myotubes overexpressing 14-3-3 g , cotransfection with GFP plamsid was routinely used (1 m g/ml). AChR clusters were detected with FITC-conjugated a -bungarotoxin (1 m g/ml, Molecular Probes). 14-3-3 g was detected with anti-14-3-3 g polyclonal antibody after permeabilization with 0.5% Triton.

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