Western Blot Analysis of Apoptosis Markers

Using published protocols [107 (link),110 (link),111 (link)], MCF7 and MCF10 cells were homogenised in RIPA lysis buffer, supplemented with a cocktail of protease inhibitors (cOmplete; Roche Diagnostics, Milano, Italy). Equal amounts of proteins were separated by 4–20% SDS-polyacrylamide gel electrophoresis (Criterion TGX Stain-free precast gels and Criterion Cell system; Bio-Rad, Hercules, CA, USA) and transferred onto nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo System. When indicated, the membranes were probed using the rabbit monoclonal anti-cleaved caspase 7 and anti-XIAP (Cell Signaling Technology) primary antibodies. After the incubation with the appropriate horseradish-peroxidase-conjugated secondary antibody (Cell Signaling Technology), bands were visualised using the Clarity Western ECL substrate with a ChemiDoc MP imaging system (Bio-Rad). To monitor for potential artefacts in loading and transfer among samples in different lanes, the blots were routinely treated with the Restore Western Blot Stripping Buffer (ThermoFisher Scientific) and re-probed with the goat anti-Lactate dehydrogenase (LDH)-A (Santa Cruz Biotechnology) and the mouse anti-vinculin primary antibodies. The stain-free gel was used as loading control as well. When appropriated, bands were quantified for densitometry using the Bio-Rad Image Lab software.