Mitochondrial Protein Folding Assay

Mitochondrial protein folding assay was carried out as previously described^{19 (link)}. LNCaP or DU145 cells were transfected with control non-targeting siRNA or LONP1 and/or ClpP-directed siRNA for 48 h. Mitochondrial fractions were prepared using a mitochondria isolation kit (BioVision Milpitas, CA, USA #K256-25) following the manufacturer’s recommendations. Briefly, cells were mechanically disrupted by 70 strokes in a Dounce homogenizer in isolation buffer A containing protease inhibitor cocktail. Cell debris and nuclei were removed by centrifugation at 700 g for 10 min, and mitochondrial fractions were precipitated by centrifugation at 3000 g for 25 min. To obtain highly enriched mitochondrial fractions, samples were subject to another round of centrifugation at 12,000 g for 10 min in isolation buffer C, and the final pellet was used as an isolated mitochondrial fraction. Mitochondrial fractions were suspended in an equal volume of mitochondrial fractionation buffer containing increasing concentrations of CHAPS (0, 1, or 2.5%). Samples were incubated for 20 min on ice, and detergent-insoluble protein aggregates were recovered by centrifugation (20,000 g) for 20 min. Pelleted proteins were separated by SDS gel electrophoresis and visualized by SYPRO Ruby staining (Invitrogen).