Mitochondria or subfraction of mitochondria was purified from rat brain as previously described.10 (link), 19 (link), 56 (link) In brief, rats (n=12) were killed and brain tissue was homogenized in isolation buffer (250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 0.5% BSA). After a series of centrifugations, the nuclear material and the mitochondrial pellet containing synaptosomes were separated. Synaptosomes were disrupted by applying 1200 psi pressure for 10 min and mitochondria were separated by ultracentrifugation. Subfractionation of mitochondria was performed as described previously.10 (link) In brief, the purified mitochondrial pellet was permeabilized and disassociated using 0.1% digitonin. Anti-VDAC (1:1000 dilution, Cell Signaling) was used as a marker for the outer membrane, and anti-COX IV (1:1000, Invitrogen) as a marker for the inner membrane to verify the quality of the fraction.