Ab188873

For immunohistochemistry, 5 μm cryosections of mouse TA muscles were fixed in 4% PFA in PBS and permeabilized with 0.1% Triton X-100 in PBS. Nonspecific binding was blocked with 5% bovine serum albumin and 0.1% Triton X-100 in PBS solution. Sections were stained overnight with rabbit polyclonal primary antibodies that recognized the C-terminal epitope of dystrophin (ab15277, 1:250, Abcam), other DAGC components (ab188873, ab189254, Abcam; sc-14176, Santa Cruz Biotechnology; all 1:500), and Alexa Fluor 488 goat anti-rabbit secondary antibody (ab150077, 1:1000, Abcam) for 1 h. Nuclei were counterstained with To-Pro dye. Antibodies and To-Pro dye were diluted in blocking buffer. Three 15 min washes with 0.1% Triton X-100 in PBS were included after each step. Section images were captured on the SP2 Leica microscope or DMI6000 Leica microscope.

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.