Western Blot Analysis of Key Proteins

Western blotting analysis was performed as previously described 12 (link). Briefly, total proteins that were extracted using a Total Cell Protein Extraction Kit (KeyGen Biotechnology, Nanjing, China) were divided into equal amounts and electrophoresed, transferred onto a nitrocellulose membrane, and blocked with 2% bovine serum albumin. Primary antibodies against MTBP (1:1000; ab115529, Abcam, Cambridge, UK), MDM2 (1:2000; ab16895), p53 (1:1000; ab131442), p21 (1:2000; ab109520), PUMA (1:2000; ab33906), active caspase3 (1:1000; ab2302), and c-myc (1:1000; ab56) were used to detect the expression of these proteins. After washing four times with TBST/0.1% Tween-20, the membranes were incubated with the corresponding secondary antibody. Bands were visualized using a chemiluminescence kit (Beyotime Biotechnology, Beijing, China) and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).