Isolation of mitochondria
Cells were suspended in mitochondria isolation buffer (250 mM sucrose,
0.1 mM EDTA, and 2 mM HEPES, pH 7.4) containing protease inhibitors and
phosphatase inhibitors (Thermo Scientific) and homogenized using 21G
needle. The homogenized cells were then centrifuged at 3,000 rpm for 10
min at 4 °C. The mitochondria-containing supernatants were transferred
to new tubes and centrifuged at 15,000 rpm for 30 min at 4 °C. After
centrifugation, the pellets (mitochondrial fraction) were lysed with
laemmli sample buffer (Bio-Rad).