L8 80m ultracentrifuge

A Mettler DCS-30 TA instrument was used to capture heating traces from 233 up to 313 K. The scanning rates were 5 K/min. Each DSC sample weighed 15–20 mg.
TEM specimens were prepared by dispersing ND suspensions in ethanol onto TEM carbon–copper grids and by drying in vacuo (10^–2 mm Hg). HRTEM imaging was performed with a JEM 2100 F transmission electron microscope with an accelerating voltage of 200 kV.
A CM-50 laboratory desktop centrifuge from ELMI Ltd. (a #50.01 rotor, 12 test-tubes of 0.2–2.0 mL, 1000–15000 rpm, RCF 15300g at r_max = 6.1 cm) and a Beckman L8-80 M ultracentrifuge (a 45Ti rotor for 6 test-tubes of 80 mL, 1000–50000 rpm) were used for fractionation.
A Kern 770 analytical balance was used for dry powder samples weighting. An Ecros 6500 shaker was used for the preparation of dispersions. A GRAD 28–35 ultrasound bath from Grad-Technology was used for preparing ND dispersions in water. A SNOL 20/300 heating oven (Snol-Term Ltd.) was used for the evaporation of ND aqueous dispersions. Automatic Eppendorf Research pipettes (Eppendorf International) were used for the preparation of calibration solutions. Polypropylene test tubes (Axygen) were used for solution preparation and storage.

CV-1 cells (in 70 to 90% confluent monolayers) in T175 flasks were infected with HPIV3 (multiplicity of infection of 0.1) in 10 ml Opti-MEM (l-glutamine and HEPES) for 90 min in a 37°C humidified, 5% CO₂ incubator. During the 90 min of incubation, flasks were gently shaken every 15 min. Viral inocula were replaced with 20 ml of complete medium with or without neuraminidase to deplete receptors for HN, and cultures were placed in a 37°C humidified, 5% CO₂ incubator. After 48 h, the cell culture supernatant fluid was collected and clarified by centrifugation (3,000 rpm for 10 min at 4°C in an Eppendorf 5810R). The clarified supernatant fluid was centrifuged (25,000 rpm for 120 min at 4°C in an SW28 rotor, Beckman L8-80M ultracentrifuge) through an 8-ml 20% (wt/vol) sucrose cushion in phosphate-buffered saline (PBS) (pH 7.4) or in PBS containing 1 mM zanamivir (pH 7.4) to prevent receptor engagement by HN. Pellets were resuspended in 200 µl of PBS with or without zanamivir (4°C, pH 7.4) for each T175 flask. Virus stocks were stored at −80°C or kept at 4°C before analysis. The titers of HPIV3 stocks were determined by a plaque assay performed as described before (24 (link)). Titers for purified viruses after storage at −80°C were 1.00 × 10⁷ PFU/ml.

LLC-PK1 cells were grown in multiple 150-mm culture dishes, and where indicated, transfected with NR or MRTF siRNA. Following 48h serum depletion, cells were washed, placed in fresh PBS, and agitated at 350 rpm at room temperature for 4min. The cell solution was subsequently spun at 3000 rpm for 10min at 4°C. The clarified supernatant was subjected to a high speed spin (40,000rpm) for 30min at 4°C using a Type 42.1 rotor and an L8-80M ultracentrifuge (Beckman). A small fraction of the isolated primary cilia was placed on a glass coverslip, dried with a stream of nitrogen, fixed and stained for acetylated tubulin to monitor the purification procedure. Cell pellets were resuspended directly in Laemmli buffer for analysis by immunoblotting, or in RIPA buffer for immunoprecipitation as described above.

ER microsome and mitochondrial isolations were performed as described previously [24 (link)]. Briefly, cells were re-suspended with iso-osmotic buffer (0.32 M sucrose, 1 mM MgCl₂, 10 mM Tris-HCl [pH 7.4]) and lysed with 20 passes using a Dounce homogenizer. The homogenate was centrifuged at 1000×g for 10 min at 4 °C, and then supernatant was removed to a new tube. The supernatant was centrifuged at 10,000×g for 30 min at 4 °C, and then supernatant was removed to another new tube. Pellet was added with PBS (mitochondria). The supernatant was centrifuged at 100,000×g for 1 h at 4 °C using an SW32.1 rotor in an L8-80 M ultracentrifuge (Beckman-Coulter) and then, supernatant was discarded. The pellets (ER fractions) were stored at -80 °C until use.