Mitochondria Isolation

Mitochondria were isolated from the skeletal muscle tissue of zebrafish by following previously published protocols (Spinazzi et al., 2012; Patra et al., 2016; Padhan et al., 2020). The tissue samples, flash frozen in liquid nitrogen and stored in a -80°C freezer, were thawed on ice. Next, the samples were incubated in isolation buffer 1 [225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA, and 30 mM Tris-HCl (pH 7.4)] and homogenized on ice using a motorized dounce homogenizer for 3 min, followed by 5 min ice incubation. The homogenization and incubation process was repeated three times. The homogenate was then collected and centrifuged at 600 × g for 5 min at 4°C. The pellets containing unbroken tissue and nuclei were discarded and the supernatant was further centrifuged at 7,000 × g for 10 min to obtain a mitochondria-containing pellet and a cytosolic supernatant. This pellet was washed with isolation buffer 2 [225 mM mannitol, 75 mM sucrose, and 30 mM Tris–HCl (pH 7.4)] and centrifuged at 10,000 × g for 10 min at 4°C. The isolated crude mitochondrial pellet was resuspended in mitochondrial resuspension buffer [250 mM mannitol, 0.5 mM EGTA, and 5 mM HEPES (pH 7.4)] and either stored for blue native PAGE (BN-PAGE) analysis or lysed for immunoblotting with mitochondria lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1% Triton X-100, and 2 mM 6-amino hexanoic acid]. The protein content was estimated by Bradford reagent.