Western Blot Analysis of DNA Damage Signaling

Whole cell and homogenized tumor tissues were lysed in NP-40 lysis buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl and 1% Nonidet P40, supplemented with Complete Protease Inhibitor Cocktail tablets and PhosStop phosphatase inhibitors (Roche, Indianapolis, IN)] and prepared as previously described (26 (link)). Total cellular proteins (40 μg) were separated on 12% or 15% SDS/PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon).. The membrane was then blocked for 1 hour at room temperature in 5% (w/v) non-fat milk in TBS-Tween-20 and probed overnight with primary antibodies followed by anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) in blocking buffer for 1 h. Membranes were subsequently incubated in Immobilon Western Blot Chemiluminescent HRP Substrate (Millipore) and developed on biomax XAR film (Kodak). Primary antibodies were purchased from Cell Signaling Technology: γH2AX (Ser139), p-Wee1 (Ser 642), Wee1, p-cdc2 (Tyr15), p-BRCA1 (Ser1524), p-Chk1 (Ser345), p-Chk1 (Ser317), Chk-1, phospho-Chk2 (Thr68), PP2A-C, PP2A-A, cleaved caspase-3 (Asp175), cleaved PARP (Asp214), p-histone H3 (Ser10), p-ATR (Ser428), and p-(Ser) 14-3-3 binding motif.