Western Blot Protein Detection

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)].