P53 do 1

The cell lysate was prepared by sonication in RIPA buffer with protease and phosphatase inhibitor cocktails. Proteins (30 µg) were separated by 8%–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, blocked in 5% milk, and incubated overnight at 4°C in primary antibodies including PARP (1:1,000; #9542, Cell Signaling, Danvers, MA, USA), E2F1 (1:1,000; #3742, Cell Signaling, Danvers, MA, USA), p53 (DO-1) (1:1,000;, cat. no. OP43, Calbiochem, San Diego, CA, USA), PUMA (1:1,000; #12450, Cell Signaling, Danvers, MA, USA), caspase 3 (1:1,000; #9662, Cell Signaling, Danvers, MA, USA), and caspase 9 (1:1,000; #9502, Cell Signaling, Danvers, MA, USA), followed by the respective anti-IgG secondary antibodies (1:3,000; Millipore, Burlington, MA, USA) for 1 h at room temperature. Membranes were developed for visualization and photography using enhanced chemiluminescence (ECL) (Millipore Corporation, Billerica, MA, USA). Optical band densities were quantified using ImageJ software, and the results were analyzed using Excel software.

The lysates (40 µg) were solubilized in Laemmli sample buffer by boiling and subjected to 10% SDS-polyacrylamide gel electrophoresis followed by electro-transfer onto a nitrocellulose filter. The membrane was incubated with a primary antibody for HOXB5 (1:500) (Thermo Fisher, Waltham, MA, USA), p53(Do-1) (1:1000), RB1(1:1000), RET(1:1000) (Cell Signaling Technology, Inc., Danvers, MA, USA). Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG was used in the secondary reaction. Immunocomplexes were visualized with an ECL Western Blot Detection System (Amersham Biosciences, Piscataway, NJ, USA). β-actin (Sigma-Aldrich, St. Louis, MO, USA) was also stained as a loading control [1 (link)].