Evaluation of Protein Expression in Muscle

Evaluation of protein expression was performed as previously described (11 (link), 28 (link)). Briefly, frozen muscle samples were homogenized in sucrose buffer pH 7.4 (10 mmol/L Tris–HCl, 1 mmol/L EDTA and 250 mmol/L sucrose), centrifuged at 760 g for 10 min at 4°C, and the supernatant was used as a total cellular protein fraction. Protein concentration was determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).
Equal amounts of protein (20–50 μg, according to the target protein) were electrophoresed, transferred to nitrocellulose membrane and immunoblotted with anti-GLUT4 (1:3500, EMD Millipore, Billerica, MA, USA, #07-1404), anti-HK2 (1:1,000, Cell Signaling Technology, Boston, MA, USA, #2867S), anti-NFKB1 (1:1000, Cell Signaling Technology, Boston, MA, USA, #12540S) or anti-RELA (1:450, Abcam, Cambridge, MA, USA, #7970). The membranes were incubated with appropriate secondary conjugated antibody, according to manufacturer's specifications, and signal was detected by enhanced chemiluminescence procedure. The optical density of the blots was quantified by densitometry (ImageScanner III, GE Healthcare, Uppsala, Sweden) and normalized by the densitometry of the respective lane measured in the Ponceau S stained membrane (29 (link)). Results were expressed as arbitrary units (AU) per μg of protein, and considering the mean of control values as 100.