Anti p27

Cells were washed with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl, 1% NP-40, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM EDTA) containing 1 mM PMSF and 1x protease inhibitor (539134, Calbiochem). Total cell lysates were then collected by sonication of the cells followed by centrifugation at 16,000g for 5 min at 4 °C. Total cell lysates (30–50 μg) were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were hybridized with anti-p53 (Proteintech Group), anti-p53-S15 (Cell Signaling Technology), p53-T18 (GeneTex, Inc), p53-T55 (Abcam), p53-T81 (Cell Signaling Technology), p53-T155 (Santa Cruz Biotechnology, Inc), anti-Cdc25B (Cell Signaling Technology), anti-Cdk1 (Santa Cruz Biotechnology, Inc), anti-pCdk1-Y15 (Abcam), anti-p27 (GeneTex, Inc), anti-p21 (Cell Signaling Technology), anti-p16 (Proteintech Group), anti-pRB (S780, Cell Signaling Technology), or anti-RB (Proteintech Group). Horseradish-peroxidase-conjugated donkey anti-rabbit (GE Healthcare Life Sciences) or anti-mouse antibodies (Proteintech Group) were used as the secondary antibodies. The T-Pro LumiLong Plus Chemiluminescent Substrate Kit (T-Pro Biotechnology) was used to visualize the antibody-bound proteins.

Total cellular proteins were extracted by lysis of the harvested cells with the protein extraction buffer at 4 °C for 1 h, followed by denaturation at 95 °C for 5 min, and then separated by regular sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) according to the procedure reported formerly [24 (link)]. Next, the proteins were transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA), after which the membrane was probed for the proteins of interest with specific antibodies. Finally, the immunoreactive bands were detected with an enhanced chemiluminescence (ECL) detection system (Amersham Life Sciences Inc., Arlington Heights, IL, USA). The expression of GAPDH was considered as a loading control. Primary and secondary antibodies used in this study includes: anti-LC3, anti-p62, anti-Beclin-1, anti-Bax, anti-Atg5, and anti-cyclin B (Cell Signaling Technology, Beverly, MA, USA); anti-p53, anti-PARP, anti-Caspase 3, anti-pro-Caspase 3, anti-cyclin A, and anti-GADPH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-p27, anti-CDK2, anti-mouse IgG, and anti-rabbit IgG (GeneTex, Irvine, CA, USA).