Lauryl maltoside

Highly purified CPI-613 and CPI-157 were synthesized from D,L lipoate as described previously
[18 (link)]. N-acetylcysteine (NAC), auranofin, resazurin, diaphorase, glutaredoxin-1, reduced glutathione, Triton X-100, digitonin, lauryl maltoside, dithiothreitol (DTT), NAD⁺, ADP, thiamine pyrophosphate, coenzyme-A (CoA), and N-ethylmaleimide (NEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Biotin-HDPD and gel filtration columns (PD10) were from Thermo Scientific (Waltham, MA, USA). 2',7'-dichlorodihydrofluorescein diacetate (DCF), dihydroethidium (DHE), and Amplex Red were from Life Technologies. Antibodies to Prx1, Prx3 and reduced lipoate were purchased from AbCam (Cambridge, MA, USA). Antibodies against dihydrolipoamide dehydrogenase (E3) were from Rockland Immunochemicals (Gilbertsville, PA, USA) and KGDH dihydrolipoamide succinyltransferase (E2) antibodies were from Cell Signaling (Danvers, MA, USA).

Complex IV activity was measured as previously described⁶³ . A buffer comprised of 10 mM potassium phosphate pH 7.0, 1 mg/ml BSA (Gold Biotechnology), and 120 mM lauryl maltoside (Sigma-Aldrich) was added to tissue homogenates and cell pellets. 2 mM cytochrome c (Sigma-Aldrich) reduced with sodium dithionite (Fisher) was added to catalyze the reaction. Measurements were taken at 550 nm at 30 s intervals for 20 min at 37 °C. Potassium cyanide (240 μM) was used to inhibit the reaction to ensure slope was specific to COX. Results were normalized to protein concentration using the DC™ Protein Assay Kit II (Bio-Rad).

Mitochondrial membranes were isolated from 2.5 × 10⁶ cells or from 200 μg of pure mitochondria as described previously (Nijtmans et al, 2002). Cells were solubilized with 3% digitonin (wt/vol) (Sigma‐Aldrich) and 0.4% (wt/vol) lauryl maltoside (Sigma‐Aldrich) or 1% digitonin (wt/vol). Pure mitochondria were solubilized with two rounds of digitonin (8 g/g and 4 g/g of protein). Ten microliters of samples were electrophoresed on a 5–13% gradient polyacrylamide gel as described previously (Nijtmans et al, 2002). Transfer of proteins onto a PVDF membrane (Bio‐Rad Laboratories) was carried out overnight at 30 V at 4°C. For second‐dimension gel electrophoresis, a lane excised from the first dimension native gel was first treated for 30 min with denaturing buffer containing 15 mM β‐mercaptoethanol and 1% SDS and then washed in 1% SDS for 1 h. The gel strip was electrophoresed on a tricine–SDS–polyacrylamide gel as described previously (Ballinger et al, 1999).
For supercomplex analysis, enriched mitochondrial fraction was solubilized with 6 g/g digitonin. 30 μg proteins were loaded to a 4–13% Bis–Tris native gel. Electrophoresis was run at 4 mA during 23 h. Proteins were transferred at 63 mA for 24 h to a PVDF membrane.

IFC-305 is the aspartate salt of adenosine prepared with adenosine-free base (MP Biomedicals, LLC, Illkirch, France) and l-aspartic acid (MP Biomedicals, Inc., Eschwege, Germany) as described (Patent No. MX220780; MX 207,422; US 8,507,459 B2). Diethylnitrosamine, sucrose, EDTA, trizma-base, KCl, MgCl₂, glutamate, ADP, succinate, 2,6-dicholorindophenol, ATP, NAD, NADH, lauryl-maltoside, cytochrome c, albumin, deoxycholate, nitrotetrazolium blue chloride (NBT), ɛ-aminocaproic acid, acrylamide, malate, potassium cyanide (KCN), antimycin A, oligomycin, rotenone, methanol, chloroform, Coomassie blue and digitonin were purchased from the Sigma Chemical Company (St. Louis, MO, USA). The oligonucleotides were manufactured by the OligoT4 Company (Irapuato, Guanajuato, Mexico).

The enzymatic activity of PPO in cell lysates was determined as an increase in a fluorescent signal upon conversion of non-fluorescent protoporphyrinogen IX to fluorescent protoporphyrin IX^{69 (link)}. Approximately 4 × 10⁶ cells were harvested in 0.06% trypsin/0.02% EDTA, washed with PBS and lysed in 100 µl PBS/1.5% lauryl maltoside (Sigma-Aldrich) supplemented with the EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). PPO activity in cell lysates was determined by incubating 10 µl of undiluted lysate with 10 µM porphyrinogen in the reaction buffer (100 mM KH₂PO₄, 0.3% (w/v) Tween-80, 5 mM DTT, 1 mM EDTA, pH 7.2) in the total volume of 20 µl in 384-well plates at 37 °C. The reactions were monitored continuously for 60 min with the fluorescence signal of the protoporphyrin IX product quantified using a CLARIOstar fluorimeter (BMG Labtech GmbH, Ortenberg, Germany) with excitation/emission wavelengths set at 410/632 nm, respectively. The data were normalized to the total protein content and analyzed using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA).