Western Blot Analysis of Cellular Proteins

Cells were collected in a conical tube and lysed in Laemmli buffer, followed by sonication and heat denaturation. Protein lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Protein-bound membranes were then incubated with blocking buffer containing 5% skim milk in Tris-buffered saline, followed by incubation with following antibodies: anti-RTA mouse monoclonal antibody α50A [13 (link)], anti-ORF57 mouse monoclonal antibody (kindly provided by Drs. Vladimir Majerciak and Zhi-Ming Zheng of National Cancer Institute, MD) [14 (link)–16 (link)], anti-c-Myc rabbit monoclonal antibody (ab32072, Abcam, Cambridge, UK), anti-Bcl-2 rabbit monoclonal antibody (Abcam), anti-Bcl-XL rabbit monoclonal antibody (Abcam), or anti-β-actin (ACTB) mouse monoclonal antibody (AC-15) (Santa Cruz Biotechnology, Inc. Dallas, TX). Membranes were subsequently incubated with anti-mouse IgG goat polyclonal antibody with HRP (Abcam) or anti-rabbit IgG goat polyclonal antibody with HRP (GE Health care, Chicago, Il). Chemiluminescent detection was conducted using a Super Signal West Pico and Femto (Thermo Fisher Scientific, Waltham, MA, USA) and a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Uncropped blot images are shown in Supplementary Fig. S1.