Bradford method assay kit

Mitochondria were isolated from adult CFW (Swiss Webster) male standard mice (Charles River Labs, Wilmington, MA), 8–16 weeks old. Mice were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg), and heparin (200 UI/kg) was used to prevent blood coagulation. Hearts were surgically removed and immediately arrested in cold (4°C) Krebs Henseleit bicarbonate buffer (KH) solution (mM): glucose 11, NaCl 118, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25 and CaCl₂ 3, pH 7.4. Myocardial sections (approximately 0.15–0.22 g) were placed in isolation buffer A (mM): sucrose 70, mannitol 210, EDTA 1 and Tris-HCl 50, pH 7.4. The tissue was finely minced and homogenized in the same Buffer A (0.1 g of tissue/ml of buffer). The homogenate was centrifuged at 3,000 rpm for 3 minutes in a Galaxy 20R centrifuge (VWR, Radnor, PA); the supernatant was centrifuged at 13,000 rpm for 10 minutes. The mitochondrial pellet was resuspended in isolation Buffer B (mM): sucrose 150, KCl 50, KH₂PO₄ 2, succinic acid 5 and Tris/HCl 20, pH 7.4). Protein concentration was estimated using the Bradford method assay kit (Bio-Rad, Hercules, CA).

cLTD was induced as previously described (Oh et al., 2006 (link)). Cultures were first incubated in ACSF for 30 min at room temperature: 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 33 mM D-glucose, and 25 mM HEPES (pH 7.3), followed by stimulation with 50 μM NMDA in ACSF (no MgCl₂) for 5 min. After NMDA treatment, neurons were replaced in regular ACSF and then subjected to the corresponding procedure at indicated time points. Cultures were then washed with ice-cold PBS once and lysed in cold 1% NP-40 homogenization buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM Na₂VO₄, and 1× Sigma protease inhibitor and phosphatase inhibitor cocktails). When indicated, cultures were pre-treated or post-treated with BAPTA-AM (20 μM), FK506 (10 μM), MG132 (10 μM), or MK801 (10 μM; all from Tocris) and were present until cells were lysed. cLTP was induced as previously described (Otmakhov et al., 2004 (link)) by application of forskolin (50 μM) and rolipram (0.1 μM; both from Tocris), 60 min before or after cLTD. Cells were lysed 60 min after cLTP or cLTD treatment. Lysates were centrifuged at 12,000 × g for 10 min at 4°C and protein in the supernatant was quantified by Bradford method assay kit (Bio-Rad Laboratories). The intensity of the cLTD protocol may vary depending on the cultures.