Phosphorimaging system

RNA extraction and Northern blotting analyses were performed as described previously (23 (link)). Briefly, total RNA was extracted using TRIzol (Thermo Fisher Scientific). 5–10 μg of RNA were separated on a denaturing formaldehyde 1% agarose gel for mRNAs and rRNAs or 10% polyacrylamide gel containing 7 m urea for tRNAs, transferred to a nylon membrane (GE Healthcare), and UV-crosslinked to the membrane. Membranes were hybridized with T7-transcribed [α-³²P]UTP- radiolabeled riboprobes. Hybridization was performed at 60 °C in 50% formamide, 7% SDS, 0.2 m NaCl, 80 mm sodium phosphate, pH 7.4, and 100 μg/ml salmon sperm DNA. Imaging and quantification were performed with a phosphorimaging system (Bio-Rad) or Typhoon imaging system (GE Healthcare). A detailed list of primers used to transcribe the riboprobes is provided in supplemental Table S2.

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).