Mitochondrial Protein Extraction and Western Blot Analysis

Proteins from cells or lungs were extracted using RIPA Lysis Buffer with Complete Protease Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). To detect the release of AIF from mitochondria, cells or tissue mitochondria isolation kit (Beyotime, Shanghai, China) were used to separate the protein, and cytoplasm (without mitochondria fractions) was collected for WB. WB was performed as described previously [23 (link)]. The protein levels were measured with specific primary antibodies including HSP70 (ab79852, Abcam, Cambridge, UK), KANK2 (PA5-116620, Invitrogen, Waltham, MA, USA), Bax (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab194583, Abcam, Cambridge, UK), GAPDH (9485, Abcam, Cambridge, UK), β-actin (ab8226, Abcam, Cambridge, UK), and AIF (4642, Cell Signaling, Danvers, MA, USA) at a dilution of 1:1000. The appropriate secondary antibodies were chosen to incubate with membrane for 2 h at room temperature. Finally, protein bands were applied with enhanced chemiluminescence detection kit (Thermo Scientific, Portsmouth, NH, USA) and imaged by fusion-capture software (Fusion FX7, Marne-la-Vallée, France).