Rotor tla 120

MCD4 cells were grown on Ø150 mm Petri dishes at 80% confluence. After treatment cells were scraped and re-suspended in General Actin buffer containing 5 mM Tris-HCl pH 8.0 and 0.2 mM CaCl₂and protease inhibitors. Cells were homogenized using a 27-gauge needle. Nuclei were removed by centrifugation at 800 x g for 10 min. Cytosol fractions were obtained by centrifugation at 150.000 x g for 1 h at 4°C in a Beckman Rotor TLA 120.1.
G-actin stock was prepared following the manufacturer’s procedures (Cytoskeleton, Inc, #AP05-A).
Cytosol (200 μg) was added in a cuvette and the fluorescence emission signal was recorded using a fluorimeter (RF-5301PC, Shimadzu Corporation, Kyoto Japan) at excitation and emission wavelengths of 370 and 430 nm, respectively. After 3 min, 100 μl of General Actin Buffer (0.4 mg/ml) was added and the fluorescent signal was acquired for the next 3 min. Formation of F-actin was initiated by a 10-fold Actin Polymerization buffer containing 100 mM Tris-HCl pH 7.5, 500 mM KCl, 20 mM MgCl₂ and 10 mM ATP and the fluorescent emission signal was recorded for 20 min.

Vesicles were prepared from rat kidney papillae or MCD4 cells. The inner medulla from rat kidney slices were excised and cut in small pieces. These pieces or MCD4 cells were homogenized manually with a mini-potter in ice-cold Isolation medium (220 mM mannitol, 70 mM sucrose, 5 mM EGTA, 1 mM EDTA, 20 mM Tris-HCl pH 7.4). Nuclei and mitochondria enriched fractions were removed by centrifugation at 8000× g for 20 min while membrane was removed by centrifugation at 17,000× g for 1 h. The supernatant was spun at 200,000× g in a Beckman Rotor TLA 120.1 for 1 h at 4 °C. The final pellet, enriched in intracellular vesicles, was gently resuspended in Isolation medium using a 30-gauge needle and used for experiments.