4.7. Protein Expression

Cells were harvested and homogenized in RIPA buffer (Sigma-Aldrich^® GmbH, St. Louis, MO, USA) containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.1% (v/v) sodium dodecyl sulfate and protease, phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentrations were determined by DC™ Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and equal amounts of proteins (25 µg) were separated by 8% and 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with antibodies specific to Myosin (1:200), MuRF-1 (1:200), and Foxo (1:200) (all from Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:10,000, Acris Antibodies GmbH, Herford, Germany), respectively, and appropriate secondary dye-conjugated antibodies goat anti-mouse IRdye800, IRdye650 (1:10,000; LI-COR^®, Lincoln, NE, USA) to reveal protein bands for 1 h at room temperature. Membranes were scanned using the Odyssey SA Imaging System (LI-COR^®, Lincoln, NE, USA). Experiments were performed in duplicates and the signal intensity was normalized to GAPDH.