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i) was determined by fitting the FP changes to the concentration of the small molecule competing ligand by using an equation developed in Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S. Anal. Biochem. 2004, 332, 261. For cytochrome c release, mitochondria were isolated from mouse liver using differential centrifugation for incubation with exogenous proteins as described by Frezza, C.; Cipolat, S.; Scorrano, L. Nat. Protoc. 2007, 2, 287. Briefly, livers were removed from euthanized animals and immersed in isolation buffer, IBc (10 mM Tris/MOPS, 1 mM EGTA/Tris, 0.2 M sucrose, pH 7.4). Samples were washed 5 times with IBc to remove excess blood and minced on ice in IBc. The tissue was then homogenized in a glass potter using a Teflon pestle for 4 strokes in fresh IBc. The homogenate was centrifuged at 600g for 10 min at 4 °C in a JA 25.4 rotor (Beckman). The supernatant was then centrifuged at 7000g for 10 min to pellet mitochondria. The pellet was washed in fresh IBc. The final pellet was resuspended in a small amount of IBc.
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