Brain acetylcholinesterase activity, thiobarbituric acid reactive species, nitrite, reduced glutathione, catalase levels, and mitochondrial complexes were also estimated.
Mitochondrial complexes were stained by Coomassie Blue G-250 on the same gel.
Mitochondrial complexes were analyzed by performing BN-PAGE on the control and diabetic mitochondria as previously described [ 15].
Mitochondrial complexes were separated on a 3 12% acrylamide blue native– polyacrylamide gel (BN-PAGE) as described elsewhere (García-Ruiz et al., 2010).
After the release of drug inhibition, mitochondrial translation resumed and the assembly of newly-synthesized mitochondrial complexes was investigated by collecting cells at different time points (0, 6, 15, 24, 48, 72, and 96 hours).
Mitochondria are dysfunctional in cancer: the activity of mitochondrial complexes is decreased, the mitochondrial generation of H2O2 is substantially decreased [10], the mitochondrial-K+ channel axis is suppressed [11], the oxidant-dependent inhibition p38 MAPK is impaired, and p53 suppresses mitochondrion-driven apoptosis [12].
Since the main part of silver stained complex IV in the gel from expressed CIV-G cells (Figure 2A) was found at the position of the endogenous complex and not at mass shifted fluorescent complex IV, it is obvious that only a minor part of inner membrane mitochondrial complexes are fluorescently tagged due to the moderate expression level of FP-subunits.
Respiration via mitochondrial complexes was evaluated using saponin-permeabilised cells or shredded tumour tissue, suspended in the mitochondrial respiration medium MiR06.
For second-dimension BN/SDS-PAGE, a lane containing mitochondrial complexes was excised from the one-dimension gel, as previously described (García-Ruiz et al., 2010).
A subunit of ATP synthase, an H+ transporting, mitochondrial complex, was identified in one spot which was influenced by coccidia infection and showed a coccidia infection×genetic line interaction (Table 3; Figure 6).
Mitochondrial respiratory complexes were measured in adult muscle tissue of 24‐month‐old pink1 −/− and wt zebrafish.
