Ab38497

p53, p53 Ser15-P, p53 Ser392-P, p21, and PCNA were examined by immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded sections after xylene deparaffinization and alcohol rehydration. Antigen retrieval was achieved by microwaving in 10 mmol/L sodium citrate buffer at pH 6 for 12 min. Endogenous peroxidase was quenched by incubating the slide with 3% H₂O₂ for 10 min. After washing, slides were incubated in saturating solution (PBS containing 1% BSA) for 30 min at room temperature (RT), followed by a 1 h incubation at RT with primary antibodies against p53 (ab4060, Abcam), p53 Ser15-P (ab38497, Abcam), p53 Ser392-P (ab33889, Abcam), p21 (#2947, Cell Signaling), and PCNA (ab15497, Abcam). After washing, slides were incubated for 30 min at RT with biotinylated anti-rabbit secondary antibody (E0432, Dako S.p.A) and for 30 min at RT with HPR-streptavidin. The peroxidase reaction was developed with 3, 3′-diaminobenzidine (Dako), and sections were counterstained with hematoxylin. Slides incubated with secondary antibody alone were used as negative controls.

Whole cell protein extracts were prepared by cell lysis with SDS-PAGE buffer (80 mM Tris-HCL pH 6.8, 16% glicerol, 4.5% SDS, 450 mM DTT, 0.01% bromophenol blue) with 200U/ml benzonase (Sigma) and 50 µM MgCl₂ and boiling for 5 min. Equal amounts of protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Immunoblotting was performed with antibodies against the following proteins: H3K36me3 (ab9050; Abcam), histone H3 (ab1791; Abcam), phospho-53BP1 (phosphoSer1778; No.2675, Cell Signaling, Danvers, MA), phospho-ATM (phosphoS1981; No.200-301-400s, Rockland, Gilbertsville, PA), total ATM (PC116; Millipore), total RPA32 (ab2175; Abcam), phospho-RPA32 (phosphoSer4/Ser8; A300-245A, Bethyl, Montgomery, TX), α-Tubulin (T5168; Sigma); γH2AX (phosphoSer139; 05-636, Millipore); Histone H2B (ab1790; Abcam); GFP (11814460001; Roche, Basel, Switzerland); total p53 (sc-263; Santa Cruz); phospho-p53 (phosphoSer15; ab38497, Abcam); p21 (sc-397; Santa Cruz).