Mitochondrial protein synthesis can be analyzed in cultured cells by metabolic labeling with [35S]methionine/cysteine in the presence of emetine or anisomycin to inhibit cytoplasmic ribosomes (Leary and Sasarman, 2009 (link); Richter et al., 2013 (link)). Typically, at least 8 of the 13 mammalian proteins can be robustly identified with this approach, but there are differences between mouse and human in the migration of individual polypeptides through SDS-PAGE (Leary and Sasarman, 2009 (link)). Pulse labeling of mitochondrial translation products in cultured cells was performed as described previously (Richter et al., 2013 (link)) with the following modifications. In the first approach, cells were pretreated with 100 µg/ml anisomycin to inhibit cytoplasmic translation and then pulsed with [35S]methionine/cysteine (EasyTag; PerkinElmer) for 15–240 min in the presence or absence of actinonin, chloramphenicol, or ethanol added directly in the pulse. In the second approach used for ATP5B siRNA, HEK293 cells were first incubated in labeling medium lacking methionine and cysteine for 25 min, and then anisomycin was added along with 50-µM puromycin for 15 min. The medium was replaced with fresh labeling medium containing anisomycin and [35S]methionine/cysteine in the presence or absence of actinonin. All samples were treated with benzonase according to the manufacturer’s instructions and then mixed with gel loading buffer (186-mM Tris-HCl, pH 6.7, 15% glycerol, 2% SDS, 0.5 mg/ml bromophenol blue, and 6% β-mercaptoethanol). A 12–20% gradient SDS-PAGE was used to separate samples, which were then dried for exposure with a phosphor screen and scanned with a Typhoon 9400 or Typhoon FLA 7000 (GE Healthcare) for quantification. Gels were rehydrated in water and Coomassie stained to confirm loading.