Mitochondrial Translation Profiling via Pulse Labeling

Pulse labeling experiments to evaluate mitochondrial translation were performed as described previously (23 (link)). Briefly, 143B cells were incubated for 20 min in methionine- and cysteine-free DMEM (Sigma) supplemented with 10% dialyzed serum and 2 mml-glutamine. Emetine dihydrochloride (100 μg/ml) was added for 5 min to inhibit cytosolic translation followed by addition of 100 μCi/ml S³⁵-labeled methionine and cysteine mixture (PerkinElmer Life Sciences). Labeling was performed for 1 h, and then cells were lysed. The protein content of each sample was measured using the Bradford assay (Bio-Rad), and 50 μg of each protein sample were resolved by 15–20% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue to confirm equal loading, then dried, and exposed. Imaging and quantification were performed with a phosphorimaging system (Bio-Rad).