Muscle samples were blotted and immediately frozen in liquid N2 before being homogenized in ice-cold lysis buffer containing protease and phosphatase inhibitors48.
The muscle samples were blotted dry and frozen in liquid nitrogen.
Two murine sera with unknown irisin levels, human serum samples with irisin levels previously measured with a corresponding ELISA kit (based on pAb-A), and a murine muscle sample were analyzed on the same blot.
Muscle samples were quickly rinsed in cold saline, blotted and immediately frozen in liquid nitrogen and stored at −80°C for later analyses.
SCP, MDP, IL, and CR performed muscle biopsy, immunohistochemistry, and Western blot on muscle samples.
Total proteins were extracted from the muscle samples and measured by Western blot analysis.
The regenerative potential defect of satellite cells was also investigated at the biochemical level; proliferating cell nuclear antigen (PCNA) and embryonic myosin heavy chain (eMHC, a known regeneration marker in the adult) were analyzed by western blot in muscle samples after injury.
The C-terminus antibody did not detect bands in the skeletal muscle samples despite equal protein loading (actin blot), perhaps due to differential affinity of the antibody in heart vs. skeletal muscle samples.
Contralateral tibialis anterior muscle samples were also used for Western blot analysis.
Features of gene-specific primer pairs are listed in Table 5.> For western blot analysis, frozen muscle samples (30 mg) were homogenized in RIPA buffer (radioimmunoprecipitation assay buffer; 50 mM Tris, 150 mM NaCl, 10% glycerol, 0.1% SDS, 1% Triton X-100, 1 mM EDTA, 0.5% deoxycholate, 1% protease inhibitor mix; pH 7.5) using an Ultraturrax (IKA Werke GmbH, Staufen, Germany).
