Subsequently 2 [micro]M rotenone (an inhibitor of complex I), 10 mM succinate (a substrate for complex II), 4 [micro]M antimycin
A (an inhibitor of complex III), and 1mM AA/100 [micro]M TMPD (AA/TMPD act as electron donors to cytochrome C in complex IV) were sequentially added.
(a) Parameters of oxidative phosphorylation were determined according to rate of oxygen consumption after addition of oligomycin, FCCP, and antimycin
Briefly, complex I assay was carried out, by incubating 15 [micro]g of freeze-thawed mitochondrial extract in 1 mL of assay medium (25 mM potassium phosphate, pH 7.4, 5 mM Mg[Cl.sub.2], 2 mM NaCN, 2.5 mg/ml bovine serum albumin, 13 mM NADH, 65 [micro]M ubiquinone, and 2 [micro]g/ml antimycin
A), and then measuring the decrease in absorbance at 340 nm due to NADH oxidation using a Cary 1E UV-visible spectrophotometer.
For the evaluation of ATP content under strict glycolytic conditions, M0, M1, and M2 were incubated for 5 hours at 37[degrees]C in both in the presence of rotenone (1 mmol/l) that of antimycin
A (1 mmol/l), .
There were no differences in OCR between treatment groups following the addition of oligomycin or rotenone and antimycin
indicating normal response to these inhibitors.
Chemical additions were prepared in 1x MAS at 10x the final concentration required (2mM ADP, 2 [micro]M oligomycin, 6 [micro]M FCCP, and 2 [micro]M antimycin
Natural astaxanthin (NA; purity > 97% HPLC, powder, Lot: 5M4707V), hydroxypropyl-[beta]-cyclodextrin (CD; DS = 0.67), MitoTEMPO (SML0737; >98% HPLC), N-acetyl-L-cysteine (NAC; A9165), and antimycin
A (Streptomyces sp.) were purchased from Sigma-Aldrich Co.
The first evidence demonstrating TFEB as a transcriptional regulator of mitophagy came from the observation that mitochondrial depolarizing agents, oligomycin and antimycin
A, induced TFEB dephosphorylation and nuclear translocation .
The sensor cartridge was loaded with oligomycin (1 [micro]M, port A), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 [micro]M for HepG2 and 0.3 [micro]M for mouse or human primary hepatocytes, port B), and rotenone plus antimycin
A (1 [micro]M each, port C) to measure the bioenergetics profile.
In short, post-synaptic GABAA signaling was studied in cerebellar stellate cells using antimycin
A, a cytochrome c reductase inhibitor that disrupts the electron transport chain in the mitochondria, leading to the cessation of adenosine triphosphate (ATP) production .
A simple titration protocol was used, including (1) ROUTINE respiration of intact cells (R,2) noncoupled respiration induced by optimum concentrations of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) corresponding to electron transfer capacity (E), and (3) antimycin
A-inhibitied respiration corresponding to residual oxygen consumption (Rox).
Many mitochondrial poisons are known complex inhibitors (e.g., rotenone and antimycin
A) and uncouplers (e.g., 2,4-dinitrophenol and carbonyl cyamide 4-(trifluoromethoxy)phenylhydrazone, or FCCP) of oxidative phosphorylation.