Ab3274

Cells were grown on semipermeable filter supports in a transwell chamber (0.4 μm pore size, Transwell Permeable Supports, 3460, Corning, NY, USA) and treated with vehicle or 10⁻⁹ M dDAVP applied to the basolateral side for 30 min without a period of dDAVP-mediated AQP2 induction. Cells were washed twice in PBS and then fixed with 4% paraformaldehyde (P2031, Biosesang, Seoul, Korea) for 30 min at room temperature. Cells were washed twice in PBS and permeabilized with 0.3% Triton X-100 (T8787, Sigma, St. Louis, MO, USA) for 15 min at room temperature. Following permeabilization, cells were incubated with anti-AQP2 antibody (1:200, AB3274, Millipore, Burlington, MA, USA) and anti-phosphorylated AQP2 at serine 256 (1:100, ab111346, Abcam, Cambridge, UK) in PBS at 4 ℃ overnight. Cells were washed and incubated with goat-anti-rabbit IgG Alexa Fluor 488 secondary antibody (A11008, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. The nuclei were stained with DAPI (D1306, Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature and cells were mounted with an antifading reagent (P36934, Invitrogen, Carlsbad, CA, USA). Immunofluorescence microscopy was carried out using a laser scanning confocal microscope (Zeiss LSM 5 EXCITER, Jena, Germany).

IMCD cells were grown to confluency on semipermeable filter supports in a transwell chamber (0.4 μm pore size, Transwell® Permeable Supports, catalog no. 3460, Corning) for 4 days. On day 5, IMCD cells were subjected to treatment with vehicle, dDAVP (10⁻⁹ M, basolateral sides only), and TAM (100 nM, both apical and basolateral sides of the cells) for 15 min and then fixed with 3% paraformaldehyde in PBS, pH 7.4 for 20 min at room temperature. After fixation, cells were washed twice in PBS and permeabilized with 0.3% Triton X-100 in PBS at room temperature for 15 min. Cells were washed and incubated with anti-AQP2 antibody (1,400, AB3274, Millipore) in PBS overnight at 4°C, and nuclei were stained with DAPI (D1306, Molecular Probes). After incubation, cells were washed with PBS and incubated with goat-anti-rabbit IgG Alexa Fluor 488 secondary antibody (A11008, Molecular Probes) for 2 h at room temperature. Cells were washed in a hydrophilic mounting medium containing antifading reagent (P36930, Molecular Probes). AQP2 immunolocalization was observed using a laser scanning confocal microscope (Zeiss LSM 5 EXCITER, Jena, Germany).

Immunoblot analyses were conducted with the experimenter blinded to the treatment received. The primary antibodies were rabbit anti-AQP2 (AB3274; Millipore; 1:600), sheep anti-NKCC2 (DSTT Dundee; 1:10 000), and rabbit anti-NCC (AB3553; Millipore; 1:1000); the secondary antibodies were horseradish peroxidase–conjugated goat antirabbit Ig (sc-2301; Santa-Cruz; 1:2000) and horseradish peroxidase–conjugated donkey antisheep Ig (A3415; Sigma; 1:20 000). Immunoblot analyses were conducted with the experimenter blinded to the treatment received (hence, the lack of systematic lane order). In the densitometry analysis, band density was divided by the time taken to collect the urine sample used for urinary extracellular vesicle preparation so that each result represents the abundance of antigen excreted per unit time.