Sw 32.1 ti

Cells were cultured on 150 mm plates, then rapidly transferred to ice where the media was removed and were washed with cold PBS. Cells were lysed (50 mm Tris, pH 7.2, 10 mm Mg (Ac)₂, 40 mm NH₄Cl 100 mm KCl, 1% DDM, 1 mm ATP, 400 μg/ml chloramphenicol and 1 mm PMSF) and were incubated on ice for 20 min. Cell lysates were clarified following centrifugation for 10 min at 20 000 × g at 4°C, then protein concentrations were measured (Bradford). From each cell lysate, a total of 1 mg of protein was loaded on top of a 16 mL linear 10–30% sucrose gradient (50 mm Tris, pH 7.2, 10 mm Mg (Ac)₂, 40 mm NH₄Cl 100 mm KCl, 1 mm ATP and 1 mm PMSF) and was centrifuged for 15 h at 4°C and 74 400 × g (Beckman SW 32.1 Ti). From the gradient, 24 equal volume fractions were collected for either protein or RNA isolation. Samples for protein analysis were precipitated with TCA. For RNA isolation, fractions were combined according to the established sedimentation profile of mitochondrial ribosomes and then were concentrated using Microsep™ centrifugal filter (MWCO 10kD) at 7500 × g for 90 min at 4°C. RNA was isolated using TRIzol LS reagent (Thermo Fisher).

Following procedures previously described (10 (link)), cells were cultured on 150-mm plates and then rapidly transferred to ice where the medium was removed, and cells were washed with cold phosphate-buffered saline. Cells were lysed [50 mM tris (pH 7.2), 10 mM Mg(Ac)₂, 40 mM NH₄Cl, 100 mM KCl, 1% dodecyl-maltoside (DDM), 1 mM adenosine triphosphate (ATP), chloramphenicol (400 μg/ml), and 1 mM phenylmethylsulfonyl fluoride (PMSF)] and incubated on ice for 20 min. Cell lysates were clarified following centrifugation for 10 min at 20,000g at 4°C, and then protein concentrations were measured (Bradford). From each cell lysate, a total of 1 mg of protein was loaded on top of a 16-ml linear 10 to 30% sucrose gradient [50 mM tris (pH 7.2), 10 mM Mg(Ac)₂, 40 mM NH₄Cl, 100 mM KCl, 1 mM ATP, and 1 mM PMSF] and centrifuged for 15 hours at 4°C and 74,400g (Beckman SW 32.1 Ti). From the gradient, 24 equal volume fractions were collected for either protein or RNA isolation. Samples for protein analysis were precipitated with trichloroacetic acid (34 (link)). For RNA isolation, fractions were combined according to the established sedimentation profile of mitochondrial ribosomes and then concentrated using a Microsep centrifugal filter with a molecular weight cutoff (MWCO) of 10 kDa at 7500g for 90 min at 4°C. RNA was isolated using TRIzol LS reagent (Thermo Fisher Scientific).

The sedimentation properties of GTPBP8 and the ribosomal proteins in sucrose gradients were analyzed as described before [36 (link)]. Cells or mitochondria were lysed in 1% DDM lysis buffer (50 mM Tris, pH 7.2, 10 mM Mg(Ac)₂, 40 mM NH₄Cl, 100 mM KCl, 1% DDM, 1 mM PMSF, 6 µl/mL Chloramphenicol, and 1 mM ATP) for 20 min on ice, then centrifuged at 20,000 ×g for 20 min at 4 °C. A supernatant containing 900 g of total protein was loaded onto a 16 mL linear 10–30% sucrose gradient (50 mM Tris, pH 7.2, 10 mM Mg(Ac)₂, 40 mM NH₄Cl, 100 mM KCl, and 1 mM PMSF) and centrifuged for 15 h at 4 °C with 74,400 ×g (SW 32.1 Ti; Beckman Coulter). 24 equal volume fractions were collected from the top and subjected to TCA precipitation. Samples were separated by SDS-PAGE for subsequent immunoblotting. For RNaseA treatments, RNaseA (ThermoFisher Scientific) was added to the cell lysate at a final concentration of 600 U/mL at the stage of protein sample preparation, and sucrose gradients were prepared as described.

C. limonL. was purchased from an organic farmer in Ehime, Japan. C. limonL. was gently squeezed manually. The juice was centrifuged at 2000× g for 40 min, and the supernatant was centrifuged at 10,000× g for 60 min. The supernatant was filtered through a 0.22 μm pore filter and centrifuged at 100,000× g for 90 min in a fixed angle rotor Type 45 Ti (Beckman Coulter Inc., Brea, CA, USA). The pellet was suspended in PBS (-) and was transferred to a 30% sucrose/D₂O cushion. After centrifugation at 100,000× g for 180 min in a swinging-bucket rotor SW 32.1 Ti (Beckman Coulter Inc., Brea, CA, USA), the nanovesicle-containing fraction was resuspended in PBS (-) and was centrifuged at 100,000× g for 90 min two times. The pellet was collected and resuspended in PBS (-) for the subsequent experiments.