Western blot analysis of SLP-2 transfected cells as well as control transfected cells was performed as described above. Mouse brain homogenates were purified using a differential centrifugation method described earlier (Rosenthal et al., 1987; Moreira et al., 2003). For isolation of mitochondria-enriched fractions, adult female CD-1 mice (n = 11) were killed, and the cerebral cortex was immediately dissected and put on ice. Tissue was manually homogenized at

+4 °C in 10 mL isolation buffer [in mm: mannitol, 225; sucrose, 75; HEPES, 5; EGTA, 1; bovine serum albumin (BSA), 1 mg/mL; protease type VIII, 0.3125 mg/mL from Sigma–Aldrich at pH 7.4] and centrifuged at 2000 g for 5 min. The pellet, including the synaptosomal

layer, was resuspended in isolation buffer now containing 0.02% (w/v) digitonin and centrifuged at 12 000 g for 10 min. The pellet without the synaptosomal Anti-diabetic Compound Library datasheet layer was resuspended in isolation buffer and centrifuged Doxorubicin datasheet at 12 000 g for 10 min. The pellet was finally resuspended in 100 μL of resuspension buffer (in mm: mannitol, 225; sucrose, 75; HEPES, 5; at pH 7.4). For isolation of synaptosome-enriched fractions, adult female CD-1 mice (n = 4) were used, and the cerebral cortex was homogenized in 1.5 mL of isolation buffer [in mm: mannitol, 215; sucrose, 75; HEPES, 20; EGTA, 1; 0.1% (w/v) fatty acid-free BSA at pH 7.2] followed by centrifugation at 1300 g for 3 min. The supernatant was removed and the resuspended pellets were again

centrifuged at 1300 g for 3 min. The two sets of supernatants were pooled, topped off with isolation buffer and centrifuged at Monoiodotyrosine 13 000 g for 10 min. The supernatant was discarded; the pellet was resuspended in isolation buffer and centrifuged at 10 000 g for 10 min. The pellet was resuspended in isolation buffer without EGTA and centrifuged at 10 000 g for 10 min. The final cell fragments containing pellet were resuspended in 50 μL of isolation buffer without EGTA. For mitochondrial respiration analyses, 0.5 mg/mL protein was added into the reaction chamber of a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK) set to +37 °C and filled with 1 mL respiration buffer (in mm: sucrose, 100; HEPES, 5; KCl, 100; KH2PO4, 2; EGTA, 10 μm; pH 7.4). Prior to application of oxidative substrates, WIN 55,212-2 (WIN; Biomol International LP, Plymouth, PA, USA; final concentration 0.05 μm), which was pre-dissolved in dimethyl sulfoxide (DMSO; stock solution 100 mm; stored at −20 °C), or DMSO alone diluted in respiration buffer (1 : 2000) were added into the reaction chamber. Thirty seconds later, pyruvate (5 mm) and malate (2.5 mm) were added concomitantly as the oxidative substrates. To determine ADP-dependent respiration (complex III activity), ADP (2.5 mm) was added.