Western blot
Cell lysates were sonicated using Virsonic 100 (1-3 watts) for 5
seconds, and then centrifuged at 15000 rpm for 20 min at 4 °C. Protein
amounts were measured using a BCA protein assay kit (Pierce). The
proteins (25 μg) were separated by SDS-PAGE, and then transferred onto
PVDF membrane. The membranes were incubated with primary antibodies for
overnight in 5 % BSA at 4 °C, followed by incubation with a secondary
antibody for 2 hrs at room temperature. A femto-chemiluminescence kit
(Thermo Fisher Scientific) was used for visualization of the signal.
Western blots were analyzed using Image Lab software (Bio-Rad). The
antibodies for eEF2 (rabbit), phospho-eEF2 (T65) (rabbit), eEF2K
(rabbit), Drp1 (rabbit), SOD2 (rabbit), HA tag (mouse), and prohibitin 1
(PHB1, rabbit) were purchased from Cell signaling. GAPDH antibody
(mouse) was purchased from Santa Cruz biotechnologies. 6 X His antibody
(mouse) was purchased from Clontech.