Pulse-Labeling of Mitochondrial Proteins

Pulse-labeling of mitochondrial protein synthesis was performed as described previously (34 (link)). Myoblasts (4.0 × 10⁵) were inoculated on 6-well plates and cultured at 37°C for 10 min with 1 ml of l-glutamine/l-cysteine-free DMEM (21013-024, Gibco) containing 10% dialyzed FBS (Gibco), 2 mM l-glutamine (Sigma) and 10 mM taurine, followed by addition of 50 μg/ml emetine to inhibit cytoplasmic protein synthesis and incubation for 10 min. Then, the cells were pulsed with 7.4 MBq (0.2 mCi) of [³⁵S] Met and [³⁵S] Cys (EXPRE³⁵S³⁵S Protein Labeling Mix, [³⁵S]-, PerkinElmer) and incubated at 37°C for 1 h to specifically label mitochondrial translation products. The medium was changed to DMEM-high glucose (D5796, Sigma-Aldrich) containing 10% dialyzed FBS (Gibco) and 10 mM taurine, and incubated for 10 min. Cell lysates (25 μg total proteins) were resolved by Tricine-SDS-PAGE (16.5%), and the gel was CBB-stained (CBB stain one, #04543-51, Nacalai Tesque) and dried on a gel drier (AE-3750 RapiDry, ATTO). The dried gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) to visualize the radiolabeled bands using the FLA-7000 fluorimager (Fujifilm).