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At a given time, aliquots were taken for 15% SDS-PAGE and ATP amount analysis.
YKX1 grew faster than Y-94 in the same medium measured by ATP amount (Figure 2).
ATP amount was monitored with rLuciferase/Luciferin chemiluminescence-based ATP detection kit (Promega) according to the manufacturer's instructions.
Viability was quantified 72 h post-siRNA transfection by measuring total ATP amount with the luminescent-based CellTiterGlo (CTG; Promega) assay in a 384-well format.
After centrifugation (10,000g for 3 minutes at 4°C), the ATP amount in the supernatants was quantified by a luciferin/luciferase assay (FluoProbes, Interchim, Montluçon Cedex, France) using a portable tube luminometer (Junior LB 9509, Berthold Technologies, Bad Wildbad, Germany) according to the instruction manual.
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ATP amounts were calculated from a log log graph generated for the ATP standard using Magellan software (Rodenberg, Germany).
Analysis of relative mitochondrial ATP amounts using COX-8 luciferevealedealed that the mitochondrial ATP level was significantly reduced in LETM1 overexpressed cells compared to control (Fig. 1C), indicating that LETM1 inhibited the production of mitochondrial ATP.
(C ) Relative ATP amounts.
ATP amounts were normalized to protein and presented as percent relative to control.
While excretion of acetate would yield even higher ATP amounts, it would simultaneously give rise to higher acid export, which is restricted in the model.
Although considerable ATP amounts are needed for lipid synthesis, amino acid transporters, upper glycolysis and biomass, the high (unspecific) ATP consumption through reaction r77 (966 μmol/gDW/h) indicates that there seems to be an overload with ATP which is either used for certain maintenance processes or passed through futile cycles.
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