Ja 20

Initially, 50 mL of first-morning urine was collected from all subjects in sterile containers. All urine samples were processed within 1 h. Urinary cells were removed by centrifugation at 300 ×g (Rotor: JA-20; Beckman Coulter, Fullerton, CA, USA) for 15 min at 4°C, followed by centrifugation at 17,000 ×g (Rotor: JA-20; Beckman Coulter, Fullerton, CA, USA) for 15 min at 4°C and ultracentrifugation at 170,000 ×g for 70 min at 4°C (Rotor: SW 32Ti; Beckman Coulter, Brea, CA, USA). Pellets were washed using 8 mL of sterile phosphate-buffered saline (PBS) and ultracentrifuged at 170,000 ×g for 70 min. Subsequently, pellets were suspended in 100 μL of PBS and were stored at −80°C for further analyses.

Initially, 100 mL of first-morning urine was collected from all subjects in sterile containers. All the samples were processed within 1 h of collection. Urinary cells were removed by centrifugation at 2000 ×g (Rotor: JA-20; Beckman Coulter, Fullerton, CA, USA) for 15 min at 4°C and at 10,000 ×g for 15 min at 4°C. Subsequently, the supernatant was ultracentrifuged at 170,000 ×g for 70 min at 4°C (Rotor: SW 32Ti; Beckman Coulter, Brea, CA, USA). After removing the supernatant, the pellets were washed with 8 mL of sterile phosphate-buffered saline and ultracentrifuged at 170,000 ×g for 70 min. The pellets were then suspended in 100 μL of phosphate-buffered saline and stored at −80°C for further analyses.

Plasma membrane-enriched vesicles were isolated by discontinuous sucrose gradient centrifugation, according to the method of Wang et al. (2001 (link)) and Mandala and Taiz (1985 (link)) with modifications. Roots were washed with cold deionized water and homogenized in extraction medium containing 50 mM Tricine-Tris (pH 7.8), 3 mM EGTA, 3 mM MgSO₄, 0.5% (V/V) PVP, 2 mM DTT, 0.2 mM PMSF, 5% (V/V) glycerol, and mannitol, which produced the same osmotic potential as within leaves. Two milliliters of the medium was used for each 2 g of fresh material. The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000 g for 20 min (Beckman JA-20). The supernatant was loaded on a 0%: 36%: 45% (W/W) sucrose gradient solution (5 mM HEPES adjusted to pH 7.5 with Tris, and 1 mM DTT) and centrifuged at 100,000 g for 2 h (Beckman SW40Ti). The plasma membrane-enriched vesicles were located at the 36%: 45% (W/W) sucrose interface. These vesicles were carefully collected and diluted 3–4-fold with dilution buffer (3 mM MgSO₄, 10 mM HEPES–Tris pH 7.5, 1 mM DTT) and centrifuged at 100,000 g for 30 min (Beckman type 65). The pellets were suspended in a storage buffer (40% (V/V) glycerol, 2 mM DTT, 10 mM HEPES, adjusted to pH 7.0 with KOH).