Optima le 80k ultracentrifuge

Lentivirus particles were generated by three plasmid expression system, in which HEK 293T cells were co-transfected with the following three vectors: packaging GAG- pol (Addgene), envelope pCMV-VSV-G (Addgene), and TET1-PSF Lenti or PSF lenti vector. One day before the transfection, HEK-293T cells were plated to be 60% confluent. On the next day, cells were fed with fresh medium and transfected using MIRUS (TransfectionExperts, MIR2300) transfection reagent according to manufacturer instructions. Briefly, 2.2 µg packaging GAG-Pol, 1.2 µg Envelop VSVG, and 5 µg of each TET1-PSF-lenti and PSF-lenti vector were transferred to tubes containing 21 µl MIRUS mixed with 2ml serum free medium. After 15 min incubation at RT the mixture was dropped on cell culture media. 24 hrs after transfection, cell culture media was changed with a fresh media. 2 and 3 days after transfection, cell culture media containing the viral particles was collected and centrifuged for 10 min at 53000 rpm to get rid cellular debris. Finally, the collected virus containing media were centrifuged at 40000 rpm for 2 hrs using BECKMAN COULTER (OptimaTM LE-80K Ultracentrifuge). After centrifugation, most of the media was removed and the viral particles were then resuspended and filtered using 0.45 μm filters (JET BIOFIL).

Cells were grown in 70 ml SDC-leu medium at 30°C to an OD₆₀₀ of ~0.5–0.7 (log-phase). Cycloheximide (CHX) was added to 50 ml culture in a final concentration of 100 µg/ml, and cells were incubated on ice for 5 min. After harvesting, cells were resuspended in lysis buffer containing 10 mM HCl-Tris (pH 7.5), 100 mM NaCl, 30 mM MgCl₂ and 100 µg/ml CHX. After mechanical cell lysis using glass beads, 7 A₂₆₀ units of the cell extracts were loaded onto 5–35% sucrose gradients containing 50 mM HCl-Tris (pH 7.5), 50 mM NaCl and 10 mM MgCl₂ and centrifuged at 38,000 rpm at 4°C for 2 h 45 min using a Beckman Optima^TM LE-80 K Ultracentrifuge. Gradients were analysed using a UA-6 system (Teledyne Isco) with continuous monitoring at A₂₅₄ nm.

Cytoplasmic lysates from frozen mouse tissues were prepared as described previously (Lunelli et al., 2016 ). Cleared supernatants were loaded on a linear 15%–50% sucrose gradient and ultracentrifuged in a SW41Ti rotor (Beckman) for 1 hr and 40 min at 180,000 g at 4°C in a Beckman Optima LE-80K Ultracentrifuge. After ultracentrifugation, gradients were fractionated in 1 mL volume fractions with continuous monitoring absorbance at 254 nm using an ISCO UA-6 UV detector.

Transiently transfected U2OS-DAT cells were treated with 5 mM sodium butyrate 24 h post-transfection to enhance protein expression. U2OS-DAT cells were grown to 50–70% confluence in 10 cm plates and harvested 48 h post-transfection by scraping in PBS and pelleted by centrifugation for 5 min at 1400×g. Non-transfected U2OS cells were grown to 90% confluence prior to scraping in PBS and centrifugation. Pellets were suspended in ice-cold Tris buffer (50 mM Tris–HCl, pH 7.4) and homogenized using an Ultra Turrax homogenizer (IKA-Werke GmbH & Co.KG, Staufen, Germany). Membranes were separated from the cytosolic fraction by centrifugation at 31,000×g using an Optima LE-80K Ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) for 20 min at 4 °C. Pellets were suspended in ice-cold Tris buffer, homogenized and centrifuged once more. Final pellets were suspended in ice-cold Tris buffer, aliquoted and stored at −80 °C. Protein amount of the membranes was determined using a bicinchoninic acid protein determination^{31 (link)}.