Western blotting was executed according to well-established procedures in our laboratory [43 (link), 83 (link), 46 (link), 65 (link), 44 (link), 35 (link)]. Equal amounts of mitochondrial or cytosolic protein per lane were electrophoresed on Criterion SDS-polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore). After blocking, samples were probed with primary antibodies against cyclophilin D (CypD; AbCam, 1:12,000, 5 μg), SIRT3 (1:12,000, 5 μg), acetylated-lysine (1:1,000, 20 μg), LC3B (1:1,000, 20 μg), and Drp1 (Santa Cruz, 1:1,000, 20 μg). Proteins were visualized using enhanced chemiluminescence (GE Amersham) and densitometry performed using ImageJ. SYPRO Ruby staining (Invitrogen, Grand Island, NY) of the membrane was performed and visually inspected for consistency of protein loading [70 (link)]. All antibodies were purchased from Cell Signaling unless otherwise specified. Groups studied for western blotting were adult sham (n=3), adult I/R (n=5), MO OVX sham (n=4), and MO OVX I/R (n=6).