4.9. Preparation of Cytosolic and Nuclear Extracts

Gastrocnemius muscle tissues were minced with a tissue homogenizer in hypotonic lysis buffer [25 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM MgCl₂, and 5 mM DTT] containing protease inhibitors (1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 200 mM Na₃VO₄, 500 mM NaF, and 100 mM PMSF) for 1 min and incubated for 15 min on ice. NP-40 (2.5%) was added and the cells were lysed for an additional 10 min. Nuclei were collected by centrifugation at 7500× g for 15 min at 4 °C. The supernatant was collected as the cytosolic fraction. Nuclear proteins were resuspended in extraction buffer [10 mM HEPES (pH 7.9), 100 mM NaCl, 1.5 mM MgCl₂, 0.1 mM EDTA, and 0.2 mM DTT] and incubated for 20 min at 4 °C. The extracts were centrifuged at 16,000× g for 10 min, and the protein levels were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) according to the manufacturer’s instructions. The supernatant was used for the Western blot analysis.