Western blot analysis

RIPA buffer (Sigma-Aldrich; EMD Millipore) was utilized to extract protein from the cells at 48 h post-transfection following the standard protocol. The lysates were centrifuged at 13,000 g for 15 min at 4°C to collect the upper supernatant. The bicinchoninic acid method was utilized to measure the protein concentration. SDS-polyacrylamide gels (12.5%; Invitrogen; Thermo Fisher Scientific, Inc.) were utilized to electrophorese 30 μg of the extracted protein, which were then blotted onto polyvinylidene difluoride membranes (EMD Millipore, Bedford), followed by blocking with 5% non-fat milk. Specific primary antibodies against p53 (cat. no. 9282T; 1:5,000; Cell Signaling Technology, Inc., Beverly, MA, USA) and against β-actin (cat. no. 4967S; 1:80,000, Sigma; EMD Millipore) were added for incubation with the membrane for 12 h at 4°C, and TBST buffer was utilized to wash the membrane three times, following which corresponding horseradish peroxidase-labeled mouse IgG secondary antibody (1:1,000; cat. no. HAF007; R&D Systems, Inc., Minneapolis, MN, USA) was added for incubation with the membrane for 60 min at 4°C. The chemiluminescent reagents (GE Healthcare Life Sciences) and X-ray films (Denville Scientific, Holliston, MA, USA) were used to visualize the protein bands in accordance with the manufacturer's protocol. All assays were performed three times.