Anti p53 s15

Proteins were separated with SDS-PAGE and transferred to PVDF membrane. The following antibodies were incubated with membrane: anti-ATM (Abcam), anti-ATM-pS1981 (Abcam), anti-ATR (Abcam), anti-ATR-pS428 (Abcam), anti-DNA-PKcs-pS2056 (Abcam), anti-p53(Santa Cruz), anti-p21(Santa Cruz), anti-cleaved PARP (Cell Signaling Technology), anti-p53-S15 (Cell Signaling Technology), anti-JNK (Cell Signaling Technology), anti-phosphor-JNK (Cell Signaling Technology), anti-GAPDH (Proteintech), anti-γH₂AX (Cell Signaling Technology). HRP-conjugated anti-rabbit or anti-mouse (KPL, Inc) were then used.

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.