Preparation of mitochondrial extracts
Mitochondria were prepared as described by Abou-Khalil et al. (1985)
(1) 5×108 cells were washed once with 10 ml Grinding medium and collected by centrifugation for 5 min at 800g at 4℃.
(Grinding medium : sucrose 250 mM, EDTA 2 mM, BSA 1 mg/ml, pH 7.4)
(2) The pellet was re-suspended in 1ml of Grinding medium and sonicated.
(3) Nuclei were collected by centrifugation for 12 min at 800g at 4℃ and the nuclear proteins were extracted as previously described.
(4) The supernatant was immediately centrifuged for 20min at 8,500g at 4℃. The supernatant obtained contains the cytosolic fraction.
(5) The pellet was re-suspended with 100 l of Buffer S, sonicated and then precipitated by centrifugation for 20 min at 10,000 rcf at 4℃.
(Buffer S: sucrose 150 mM, KCl 40 mM, Tris/HCl 25 mM, BSA 1 mg/ml, pH 7.4)
(6) Protein concentration in nuclear, cytoplasmic, and mitochondrial fractions were determined according to the Bradford method.
Preparation of cytoplasmatic and nuclear extract
(1) Cells (107) were washed once with PBS and re-suspended in 500 l of Hypotonic lysis buffer A. (Hypotonic lysis buffer A: 10 mM HEPES, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA, 0.1mM DTT, 5mM PMSF, pH 7.9)
(1)After 10min, nuclei were collected by centrifugation for 10 min at 500 rcf at 4℃ in a microcentrifuge. The supernatant contains the cytoplasmic fraction.
(2)Then, the nuclear proteins were extracted with 500l of Buffer B.
(Buffer B: 10 mM HEPES, 100 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 5 mM PMSF, pH 7.9).
(3)After incubating for 20 min at 4℃, samples were centrifuged at 10,000 rcf at 4℃ for 20 min.
(4)The nuclear extracts were then quantiﬁed for protein levels according to the method of Bradford (1976) and used immediately for Western blot or kept at -80℃.