Western Blot Analysis of Apoptosis Markers

The cells were harvested, washed in cold PBS, resuspended in lysis buffer (25 mM HEPES [pH 7.4], 100 mM EDTA, 5 mM MgCl₂, 0.1 mM DTT, and protease inhibitor mixture), and sonicated to prepare cell lysates. Proteins (35 μg) present in the cell lysates were separated by performing electrophoresis on 10%–15% SDS polyacrylamide gels and were transferred onto nitrocellulose membranes, and analyzed by western blotting as described previously [42 (link)]. Immunoblotting was performed using antibodies against cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-8 (BD Pharmingen, USA), Bax and p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), DR4, DR5, and ß-actin (Sigma-Aldrich). Images were obtained using Fusion-FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France).