Nuclear extracts were prepared from 50 mg of soleus muscle using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, United States). Complete Protease Inhibitor Cocktail (Santa Cruz), Phosphatase Inhibitor Cocktail B (Santa Cruz), PMSF (1 mM), aprotinin (10 μg/ml), leupeptin (10 μg/ml), and pepstatin A (10 μg/ml) were used to maintain extract integrity and function. Nuclear extracts were dialyzed by means of Amicon Ultra-0.5 centrifuge filters (Millipore, United States).
The protein content of all samples was quantified twice using a Quick Start Bradford Protein Assay (Bio-Rad Laboratories) in order to calculate the optimal sample value for electrophoretic gel. The supernatant fluid was diluted with 2× sample buffer (5.4 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and stored at −85°C for immunoblot procedures. The quality of nuclear and cytoplasmic fractions separation was evaluated by performing GAPDH immunoblot detection in nuclear samples and lamin B1 immunoblot detection in cytoplasmic samples: no bands were detected in each case.
The protein content of all samples was quantified twice using a Quick Start Bradford Protein Assay (Bio-Rad Laboratories) in order to calculate the optimal sample value for electrophoretic gel. The supernatant fluid was diluted with 2× sample buffer (5.4 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and stored at −85°C for immunoblot procedures. The quality of nuclear and cytoplasmic fractions separation was evaluated by performing GAPDH immunoblot detection in nuclear samples and lamin B1 immunoblot detection in cytoplasmic samples: no bands were detected in each case.