Fbx32

The cell lysates were prepared in lysis buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100) at 4 °C. The extractions were separated via SDS-PAGE, transferred onto a polyvinylidine difluoride membrane (Millipore, Bedford, MA, USA), and detected using antibodies against H3, p-NFκB.p65 (S536) (Cell Signaling Technology, Danvers, MA, USA), α-actinin (ACTN), p53, p21, Cyclin D1, H3P, MYH, Twist, COX-2, ATF-3, NFκB-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), γH2A.X, myogenin, MuRF1, and Fbx32 (Abcam, Cambridge, UK). The membranes were incubated first with primary antibodies against proteins of interest and then with HRP-conjugated secondary antibodies (anti-mouse IgG, AP192P and anti-rabbit IgG, AP132P, Merck-Millipore, Burlington, MA, USA). The immunoreactive proteins were detected using ECL^TM Western Blotting Detection Reagent and Amersham Hyperfilm^TM ECL (GE Healthcare, Waukesha, WI, USA). The procedural details have been described in our previous publications [47 (link),48 (link)].

Equal amounts of total tissue lysates were separated on either 4-20%, 30 μl Mini-PROTEAN TGX™ gels (Bio-Rad Laboratories, USA) or 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were then blocked with a solution containing 10 mM Tris-buffered saline, 0.05% Tween 20, and 5% nonfat dry milk or 5% bovine serum albumin (BSA) (Sigma-Aldrich) and then incubated with primary antibodies including PGC-1α (Santa Cruz, sc-13067, dilution 1 : 500), Akt 1/2/3, (Santa Cruz, sc-8312, dilution 1 : 500), P-Akt 1/2/3 (Ser⁴⁷³) (Santa Cruz, sc-7985-R, dilution 1 : 500), FoxO3a (Cell Signaling, 2497, dilution 1 : 500), P-FoxO3a (Abcam, ab154786, dilution 1 : 500), Fbx32 (Abcam, ab168372, dilution 1 : 1000), and β-tubulin (Cell Signaling, 2146, dilution 1 : 500) over night at 4°C. The membranes were treated with secondary anti-rabbit and anti-mouse antibody (dilution 1 : 20000) for 1 h at room temperature. Following treatment with the appropriate secondary antibody, the bands were visualized using ImageQuant LAS 500 (GE Healthcare). The changes in the protein level were quantified by a densitometric method using the LASImage software. β-Tubulin was used as a lane loading control. The immunoblotting was performed at least two times.