Western Blot Analysis of Apoptosis Markers

Cells were harvested and lysed in RIPA buffer with Complete Protease Inhibitor Cocktail, EDTA-Free (Roche, Basel, Switzerland) inside. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and Western blot analyses were performed as previously described [28 (link)]. PARP-1, caspase-3 (Cell Signaling Technology, Leiden, The Netherlands), DR4 (Novus Biologicals, Abingdon, UK), DR5, P21, P53 (abcam, Cambridge, UK) primary antibodies were used. Anti-γ-Tubulin or anti-vinculin (Sigma Aldrich, St Louis, MO, USA) was used for confirmation of equal loading of proteins. Polyclonal rabbit anti-mouse immunoglobulins/HRP and polyclonal goat anti-rabbit immunoglobulins/HRP (Dako, Glostrup, Denmark) were used as the secondary antibody.