Protease cocktail tablet

The homogenized ipsilateral brain hemispheres from all groups were lysed in RIPA buffer (Thermo Scientific, USA) with one protease cocktail tablet (Roche, USA) and phosphatase inhibitors II and III (Sigma, USA). Whole lysates were normalized using the BCA assay (Thermo Scientific, USA). Fifty micrograms of each lysate was subjected to 10% SDS-PAGE and Western blot assays. The information on the antibodies used in the experiments includes: anti-total AKT (1:2000), anti-phospho-AKT Thr 308 (1:1500), phospho-AKT Ser473 (1:1500), anti-total ERK1/2 (1:2000), anti-phospho ERK Thr202/Tyr204 (1:1500), anti-Bcl2 (1:1000), anti-Bcl-xL (1:1000), anti-Bax (1:1000) (cell Signaling, USA), and GAPDH (1:3000, Santacruz, USA). Levels of phosphor-AKT and Bcl-xL were calculated with normalized intensity with GAPDH using the ImageJ program.

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.