Anti rela

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.

Whole cell extracts (WCE) were obtained by lysis with 20 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA supplemented with 0,5% Triton X-100 (Sigma-Aldrich), protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail (Thermo Scientific), 1 mM phenyl-methyl-sulfonyl fluoride (PMSF; Sigma-Aldrich), 1 μM okadaic acid (Sigma-Aldrich), dithiothreitol (DTT; Fluka). Twenty to 50 μg of WCE were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with the following primary antibodies: anti-CK2α (kindly provided by Dr S. Sarno, University of Padua, Italy); anti-CDC37 (Santa Cruz Biotechnology, Santa Cruz, CA) anti-phospho-Ser13 CDC37 (Abcam); anti-CK2β (BD Biosciences, USA); anti-phospho-Ser209 CK2β (Assay Biotech) and anti-GAPDH (Ambion); anti-RelA (Abcam) and anti-phospho-Ser529 RelA (Santa Cruz Biotechnology, Santa Cruz, CA). As secondary antibodies: anti-rabbit IgG HRP-linked antibody (Cell Signaling, Beverly, MA); HRP labeled goat anti-mouse IgG (KPL, Gaithersburg, MD, USA). Detection was performed using ECL (Pierce, Thermo Scientific), LiteAblot Extend Long Lasting Chemiluminescent Substrate (Euroclone) or LiteAblot Turbo Extra Sensitive Chemiluminescent Substrate (Euroclone) according to manufacturer's instructions.