SDS-PAGE and Western blot analysis.

Fresh or frozen muscle samples were rapidly processed to homogenize at room temperature in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 2% SDS and 3% 2-mercaptoethanol, pH 8.8, using a high-speed mechanical homogenizer (Pro 250; Pro Scientific) with three blends of 5 s each to extract total proteins. The homogenized SDS-gel samples were stored on ice for no more than 15 min before heating at 80°C for 5 min. The samples were then clarified by centrifugation at 14,000 g in a microcentrifuge at room temperature for 5 min.

The protein samples were resolved on 14% SDS-gel with an acrylamide-to-bisacrylamide ratio of 180:1 made in a modified Laemmli buffer system, in which both stacking and resolving gels were in pH 8.8 buffers. The use of pH 8.8 buffer in the stacking gel does not have notable impact on the stacking effect of discontinuous electrophoresis that is based on glycine’s slower mobility, yielding reproducible high-resolution results as shown in numerous publications of our laboratory including those cited in the present paper. On the other hand, the use of the same buffer in stacking and resolving gels simplifies the system and gives stability for bulk-casted SDS-gels to be stored at 4°C for several weeks by eliminating the diffusion between different buffers in the two gel layers as in the traditional Laemmli gel. Protein bands resolved in the SDS-gel were visualized by staining with Coomassie Blue R 250. Total protein input in each lane was quantified by ImageJ software to normalize the amount of sample loading.

The resolved protein bands in duplicate SDS-gels were electrophoretically transferred to nitrocellulose membranes using a Bio-Rad semidry electrical transfer device at 5 mA/cm² for 15 min. The blotted membranes were blocked in 1% bovine serum albumin (BSA) in Tris-buffered saline (TBS; 150 mM NaCl and 50 mM Tris, pH 7.5) with shaking at room temperature for 30 min. The blocked membrane was probed with anti-CAIII monoclonal antibody (mAb) (CP3; 26), diluted in TBS containing 0.1% BSA, with gentle rocking at 4°C overnight. The membrane was then washed three times with TBS containing 0.5% Triton X-100 and 0.05% SDS, incubated with alkaline phosphatase-labeled goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnology), washed again as above, and developed in 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution to visualize the protein bands detected by the primary antibody.