Horseradish peroxidase coupled anti rabbit or anti mouse secondary antibodies

Immunoblotting analysis was performed as described previously [19 (link)]. Whole-cell extracts (50–100 µg protein) were prepared and resolved on SDS-PAGE (XCell SureLock Mini-Cell and NuPAGE Bis-Tris Precast Gel, Thermo Fisher Scientific). Gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked, and incubated with primary antibodies for 16 h. Membranes were washed and incubated with HRP-coupled secondary antibodies. The proteins were detected via chemiluminescence using the ECL Select Western Blotting Detection Reagent (Cytiva, Tokyo, Japan). Chemiluminescence measurements were performed using an ImageQuant LAS4000 system (FUJIFILM, Tokyo, Japan). The following antibodies were used: anti-DNA-PKcs (E6U3A), anti-caspase-3, anti-DNA Ligase IV (D5N5N), anti-ATM antibody (D2M2), anti-phospho-ATM-Ser1981 (D25E5), anti-PARP, anti-phospho-p53-Ser 15, anti-p21 (12D1) antibody (Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-β-Actin (20–33) (Sigma-Aldrich; 1:4000), anti-cyclin B (BD Transduction Laboratories, Franklin Lakes NJ, USA; 1:1000), anti-tubulin (B-5-1-2) (Sigma-Aldrich; 1:4000), anti-N-Myc (B8.4B), anti-p53 (DO-1) (Santa Cruz Biotechnology; 1:500, Dallas, TX, USA), and horseradish peroxidase-coupled anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology; 1:2000).

Total protein extracts from rapamycin-treated and untreated (DMSO) cells were obtained at passages when untreated cells entered senescence and stop proliferating and at passages when rapamycin-treated cells entered into proliferative arrest using Ripa Buffer (Sigma, cat. R0278) containing protease and phosphatase inhibitor cocktails (Sigma Aldrich, cat. P8340 and P5726). Equal amounts (20μg) of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were then blocked and incubated with the following primary antibodies: anti-P16^INK4A (Abcam, ab108349), anti-phospho-RPS6^S240/244 (Cell Signaling Technology, #5364), and ß-actin (Sigma, A2228) for loading control. Detection was performed using horseradish peroxidase-coupled anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology, #7074 and #7076), ECL substrate (GE Healthcare, cat. RPN2236), and the ChemiDoc MP Imaging System (Bio-Rad). The intensity of the bands was determined by densitometry using The Image Lab Software (Bio-Rad). The immunoblotting experiments were repeated more than three times and similar results were observed.