Protein Extraction and Western Blotting

Total proteins were isolated from cells incubated with RTV, LPV or DMSO using Lysis Buffer (NP40 Cell Lysis Buffer; Invitrogen^TM, Carlsbad, CA, USA) combined with a protease inhibitor cocktail 100× (1× final; Protease Inhibitor Cocktail Set I, Calbiochem^®, EMD Chemicals Inc., Merck KGaA, Darmstadt, Germany) and a phosphatase inhibitor cocktail 50× (1× final; Phosphatase Inhibitor Cocktail 50× Set V, Calbiochem^®, EMD Chemicals Inc., Merck KGaA, Darmstadt, Germany). Proteins (20 µg) were loaded on 4–15% gradient gel (Mini-PROTEAN^® TGX^TM Precast Gels, BIORAD^®, Hercules, CA, USA), and were then transferred on nitrocellulose membrane which was blocked with TBS 1×-Milk 5%-Tween 0.1% solution. The resulting proteins blots were probed with anti-MLN64, anti-P450SCC, anti-HSD3B1, anti-OPA1, anti-Mfn2, anti-GRP78, monoclonal mouse anti-actine or anti-vinculine antibodies (references and concentration in Table 1) [11 (link),17 (link)]. Actine or vinculine were used as loading control. Addition of secondary goat anti-mouse antibody conjugated with DyLight 680 (1/15,000, #35518 Thermo Fisher Scientific, Waltham, MA, USA) or secondary goat anti-rabbit antibody conjugated with DyLight800 4× PEG (1/15,000, SA5-35571 Thermo Fischer Scientific, Waltham, MA, USA) allowed blots revelation using Odyssey infrared fluorescent system (LI-COR).