Anti porin antibody

Mitochondria were extracted as described earlier [56 (link)]. A 50 μL aliquot was sonicated and used for the measurement of protein content or Western blotting; the remaining part was stored at −80°C until the use. To confirm the presence of mitochondrial proteins in the extracts, 10 μg of each sonicated sample was subjected to SDS-PAGE and probed with an anti-porin antibody (Abcam; data not shown). The electron flux from Complex I to Complex III was measured on 10 μg of non-sonicated mitochondrial extracts, re-suspended in 150 μL buffer A (5 mmol/L KH₂PO₄, 5 mmol/L MgCl₂, 5% w/v bovine serum albumin). Then 75 μL buffer B (25% w/v saponin, 50 mmol/L KH₂PO₄, 5 mmol/L MgCl₂, 5% w/v bovine serum albumin, 0.12 mmol/L cytochrome c-oxidized form, 0.2 mmol/L NaN₃) were added for 5 min at room temperature. Each sample was incubated in the absence or presence of the Complex I inhibitor rotenone (50 μmol/L), to measure the ubiquinone-independent and the ubiquinone-dependent electron flux, respectively. The reaction was started with 0.15 mmol/L NADH and was followed for 5 min, using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments). Results were expressed as nmoles reduced cytochrome c/min/mg mitochondrial proteins.

Mitochondria were extracted as described earlier [52 (link)]. A 50 μl aliquot was sonicated and used for the measurement of protein content or Western blotting; the remaining part was stored at −80°C until use. To confirm the presence of mitochondrial proteins in the extracts, 10 μg of each sonicated sample was subjected to SDS-PAGE and probed with an anti-porin antibody (Abcam; data not shown). The electron flux from Complex I to Complex III was measured on 10 μg of not sonicated mitochondrial extracts, as reported in [52 (link)]. Each sample was incubated in the absence or presence of the Complex I inhibitor rotenone (50 μM), to measure the ubiquinone-independent and the ubiquinone-dependent electron flux, respectively. The reaction was followed for 5 min, using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT). Results were expressed as nmol reduced cytochrome c/min/mg mitochondrial proteins.

Mitochondrial fractions were isolated as previously reported [26 (link)], with minor modifications. Samples were lysed in 0.5 mL buffer A (50 mM Tris, 100 mM KCl, 5 mM MgCl₂, 1.8 mM ATP, 1 mM EDTA, pH 7.2), supplemented with protease inhibitor cocktail III (Sigma Chemical Co), 1 mM phenylmethylsulfonyl fluoride, and 250 mM NaF. Samples were clarified by centrifuging at 650× g for 2 min at 4 °C, and the supernatant was collected and centrifuged at 13,000× g for 5 min at 4 °C. This supernatant was discarded and the pellet containing mitochondria was washed in 0.5 mL buffer A and resuspended in 0.25 mL buffer B (250 mM sucrose, 15 mM K₂HPO₄, 2 mM MgCl₂, 0.5 mM EDTA, 5% w/v BSA). A 50 μL aliquot was sonicated and used for the measurement of protein content or western blotting; the remaining part was stored at −80 °C until use. To confirm the presence of mitochondrial proteins in the extracts, 10 μg of each sonicated sample were subjected to SDS-PAGE and probed with an anti-porin antibody (Abcam, Cambridge, UK; data not shown). To exclude any mitochondrial contamination in the cytosolic extracts, the absence of porin in the latter was analyzed by immunoblotting (data not shown).