Ab15895

Protein samples were extracted from cells and mitochondria, respectively, using RIPA lysis buffer (Chromotek) supplemented with protease inhibitors. Protein samples were diluted in LDS sample buffer (4×, Invitrogen) supplemented with sample reducing agent (10×, Invitrogen). Samples were heated at 95̊C for 5 min before loading on a 4–12% gradient Bis-Tris gel (Invitrogen). MOPS SDS running buffer (Invitrogen) was used and supplied with antioxidants (Invitrogen). The gel was soaked in running buffer and run at 160 V for 75 min. The gel was transferred to a 0.45 µm nitrocellulose membrane (Cytiva) and protein at 30 V for 150 min, 4̊C. The membrane was blocked for 1 h in 5% skimmed milk (Sigma) in PBS-Tween 20 (0.1%, Sigma). The membrane was probed for anti-GFP (ab183734, abcam), anti-mCherry (1C51, ab125096, abcam), anti-VDAC1 (ab15895, abcam) and anti-α tubulin (DM1α, T6199, Sigma), diluted 1 : 10 000, 1 : 3000, 1 : 1500, 1 : 1500, respectively, in 5% milk (PBS-Tween). Primary antibody signal was detected using goat anti-mouse IgG conjugated to IRDye 680RD (ab216776, abcam) and goat anti-rabbit IgG conjugated to IRDye 800CW (ab216773, abcam) secondary antibodies, diluted 1 : 15 000, imaged with an Odyssey CLx imaging system (LI-CO Biosciences).

A tissue array was used which included two tumor samples from each patient. The paraffin-embedded tissues were cut into 4 μm thick sections, then deparaffinized and dehydrated following standard procedures. Subsequently, paraffin sections were rinsed with PBS (3 × 5 min) and then blocked with 3% hydrogen peroxide at 37°C for endogenous peroxidase ablation for 10 min. Antigen retrieval was conducted by microwave heating with citrate buffer (pH 6.0) for 20 min. Then, the samples were exposed to normal goat serum at 37°C for 20 min to decrease nonspecific antibody binding. The tissue sections were incubated overnight at 4°C with the primary antibody (anti-VDAC1, 1 : 1000, ab15895, Abcam, UK; anti-Cytc, 1 : 50, sc-13561, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After rinsing in PBS, the tissue sections were incubated with horseradish peroxidase-labeled anti-rabbit antibodies at 37°C for 20 min. Then, the tissue sections were rinsed with PBS for 4 times and then dripped with freshly prepared 3,3-diaminobenzidine (DAB). Microscopically, the staining was terminated when the tissue sections were brown-yellow or brown. Subsequently, all the tissue sections were restained with hematoxylin for about 1 min. Finally, the slices were dehydrated with ethanol and toluene and then sealed with neutral gum. PBS was used to replace the primary antibody as negative control.

Left ventricular cardiomyocytes were homogenized in ice-cold lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% triton X-100, 50 mM NaF, 5 mM Na₄P₂O₇, 1 mM Na₃VO₄, 1 mM DTT, and protease inhibitor cocktail) and protein content was determined using the BCA method (Thai et al., 2018 (link)). Briefly, 30 μg of protein was separated by 4–15% SDS-PAGE and transferred onto 0.2 μm supported nitrocellulose membrane using standard protocols. Blocked membranes were exposed to primary antibodies for PINK1 (Abcam ab23707), Parkin (Santa Cruz sc-32,282), mitofusin 2 (ab56889), DRP1 (Abcam ab56788), SQSTM1/p62 (Abcam ab56416), and LC3 (MLB, M186–3). GAPDH (Abcam ab9483) or VDAC1 (Abcam ab15895) were used as loading controls. Membranes were visualized using the Licor Odyssey infrared imaging system and analyzed with ImageJ software. Band intensities of all samples were normalized to an internal housekeeping protein that was included on each gel.