Whole cell extracts were prepared using M-PER® (ThermoFisher, Waltham, MA, USA) supplemented with 1× protease inhibitor cocktail (Complete EDTA-free, Roche, Basel, Switzerland), according to the manufacturer’s instructions. Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Science, Chicago, IL, USA). Membranes were incubated with selected primary antibodies: anti-caspase 7 (9494S), anti-caspase 9 (9502S), anti-caspase 8 (9746), anti-Mcl-1 (4572S), anti-LC3B (2775), and anti-p62 (5114) from Cell Signaling (Danvers, MA, USA), anti-PARP-1 (C2-10; sc-53643) from Santa Cruz Biotechnology (Dallas, TX, USA), anti-Bcl-xL (610212) from BD Pharmingen (San Jose, CA, USA), and anti-β-actin (5441) from Sigma Aldrich (St. Louis, MO, USA).
Luminescence signals were detected with the enhanced luminol-based chemiluminescent (ECL) Plus Western Blotting Detection Systems (GE Healthcare Life Science, Chicago, IL, USA). Bands were acquired by using an Amersham Imager 600 (GE Healthcare Life Science, Chicago, IL, USA) and quantified by ImageJ 1.52a software (U.S. National Institute of Health, Bethesda, MD, USA).
Luminescence signals were detected with the enhanced luminol-based chemiluminescent (ECL) Plus Western Blotting Detection Systems (GE Healthcare Life Science, Chicago, IL, USA). Bands were acquired by using an Amersham Imager 600 (GE Healthcare Life Science, Chicago, IL, USA) and quantified by ImageJ 1.52a software (U.S. National Institute of Health, Bethesda, MD, USA).