Ab154168

Protein was extracted from muscle tissues using mechanical bead rupture in lysis buffer consisting of 9% SDS, 75 mM Tris (pH 7), 5% 2-mercaptoethanol, and HALT protease inhibitors (78425, Thermo Fisher Scientific) and normalized by tissue mass. ProteinSimple Jess was used for capillary electrophoresis along with immunodetection using anti-dystrophin (Ab154168, 1:1,000; Abcam, Cambridge, UK) and anti-αactinin (MAB8279, 1:100; R&D Systems, Minneapolis, MN, USA). A standard curve was included for each experiment to allow quantification, which consisted of ratios of C57BL/10 murine lysate diluted with mdx murine lysate from the respective muscle tissue. ProteinSimple Jess reagents were used as follows and according to the manufacturer’s protocols: 66- 440-kDa separation module (SM-W008); dystrophin was detected using the antirabbit detection module (DM-001 or 043-426 when paired with DM-002 for α-actinin detection), and α-actinin was detected with either the anti-mouse near-infrared detection module (DM-009) or the anti-mouse detection module (DM-002). A Gaussian fit was used for each peak for quantification, and the ratio of dystrophin to α-actinin was interpolated to the standard curve for each experiment.

For western blot analysis, 100 mg of muscle was collected, physically disrupted and homogenized with a pestle in the presence of silicon dioxide powder and 1 ml of lysis buffer [75 mM Tris-HCl (pH 6.8), 10% SDS, 20% glycerol, 5% mercaptan, 0.001% bromophenol blue] and incubated at 95°C for 10 min to denature proteins. Each well on a precast mini-protean TGX 4-15% gel (Bio-Rad) was loaded with 5 μg of total protein. Proteins were transferred onto a nitrocellulose membrane using a standard wet transfer in Mini Trans-Blot Cell (Bio-Rad) using the manufacturer's protocol. Nonspecific binding was blocked by incubating the membranes in 5% dry milk in PBS for 3 h. Antibodies against target proteins (ab154168 1:1000, ab15277 1:400, ab188873 1:1000, ab189254 1:1000, ab15200 1:200, Abcam; NCL-b-DG 1:200, Novocastra; sc-14176 1:200, sc-304 1:200, Santa Cruz Biotechnology) or a rabbit monoclonal anti-actin antibody (A2103 1:10,000, Sigma-Aldrich) diluted in 1% dry milk in PBS were used for protein detection. Incubation with each antibody was carried out overnight at 4°C, followed by washes and incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1/3000) for 1 h at room temperature and further washes in PBS. Enhanced chemiluminescence was used for the detection of protein bands, and images were obtained and analyzed on a C-DiGit Blot Scanner.