HEK293T (ATCC) cells were maintained in media composed of DMEM (high glucose; Biowest), sodium pyruvate (Thermo Fisher Scientific), stable glutamine (Biowest), non-essential amino acids (Thermo Fisher Scientific), 10% fetal bovine serum (EurX), and penicillin/streptomycin solution (Thermo Fisher Scientific). Cells were cultured to confluence on six 9 cm plates. Mitochondria were isolated by differential fractionation previously described42 (link) with minor adjustments. Briefly, cells were washed 2 × with ice-cold PBS. The PBS buffer was removed and the cells were scraped in ice-cold NKM buffer (1 mM Tris–HCl pH 7.4, 130 mM NaCl, 5 mM KCl, 7.5 mM MgCl) and incubated on ice for 5 min. Then 100 μl of 10xHomB buffer (225 mM mannitol, 75 mM sucrose, 10 mM HEPES–NaOH pH 7.8, 10 mM EDTA) per 1 mL of homogenate with protease and phosphatase inhibitors was added and homogenized using a Dounce homogenizer with nuclei release and integrity monitored microscopically. The nuclear fraction was separated using two rounds of centrifugation at 900 × g for 5 min at 4 °C. The supernatant was then centrifuged at 10,000 × g for 10 min to isolate crude mitochondrial pellet. The pellet was suspended in Blue Native protein extraction buffer, dosed and ATP synthase complex from 400 µg of protein was extracted with 2% Digitonin. The dimers and monomers were separated in 3–12% gradient gel followed by SDS-PAGE separation in second dimension and bands from 5–20 kDa region from Coomassie stained gel from the monomers and dimers were subjected to mass spectrometric analysis.
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