Phospho h2ax

After drug exposure, the total cellular proteins of control and treated cells were isolated using lysis buffer (distilled water containing 50 mM Tris HCL, pH 8, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.15 M sodium chloride, 0.5% NP40, 0.5 mM DTT, 1 × PhosSTOP (Phosphatase Inhibitor Cocktail Tablets, Roche, Germany), 1X Protease cocktail tablet (Roche, Mannheim, Germany), following which lysates were rotated at 4°C at 14,000 rpm for 30 min. The protein concentration was determined using the colorimetric Bradford assay (Bio-Rad) with BSA as a standard. Western blot analyses for proteins involved in different pathways, including cell proliferation, cell cycle regulatory proteins (p21, p53, cyclin B1, cyclin D), apoptosis-related agents (caspases 8 and 9), DNA damage response (DDR) pathways (Phospho-H2AX (Millipore), poly adenosine diphosphate-ribose polymerase (PARP), ATM (Gene Tex, Ridgeland, MI, USA), Phospho-ATM (Gene Tex), DNA-PKcs, Phospho-DNA-PKcs and telomere-telomerase equilibrium (hTERT [Epitomics, Burlingame, CA, USA], POT1, TRF1, TRF2). Antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), unless otherwise stated. For different protein expression profiles, the experiments were repeated between two to six times.

Western blot analyses were performed as follows. Cells were plated in 100mm dishes and treated at a confluency of 50-70%. SINE inhibitors and leptomycin B were used at the specified concentrations. Treatments were carried out for 12, 24, and 48h, as specified. Post treatment, total protein was isolated and ten micrograms were used for electrophoresis and blotted on PVDF membrane. Primary antibodies were diluted in blocking buffer (either 5% milk or 5% BSA as per antibody specifications) to a 1:1000 dilution. Secondary antibodies for housekeeping proteins such as vinculin and actin, used as internal controls, were diluted at 1:4000. Blots were developed using ECL (GE Healthcare) or Femto (Pierce Biotechnology). Primary antibodies were purchased from the following source; XPO 1 (Santacruz), p53 (Calbiochem), cleaved PARP (Cell Signaling Technologies), phospho ATM (Rockland), Exportin5 (Epitomics), phospho H2AX (Millipore).