Ab111346

Antibodies anti‐NKCC2 were from Merck Millipore (AB3562P, Merck Millipore, Burlington, MA). The antibody against the phosphorylated threonine 96 and 101 NKCC2
¹⁹ (link) was kindly provided by Prof. Biff Forbush, Yale University. Antibodies anti‐NCC were from StressMarq Biosciences Inc. (SPC‐402D, StressMarq Biosciences Inc., Victoria, BC, Canada). Antibodies against the phosphorylated Thr 53 NCC were from Phosphosolutions (p1311‐53, Phosphosolutions, Aurora, CO). The antibody against human AQP2 was previously described.
²⁰ (link) Antibodies against the phosphorylated 256 AQP2 were from Abcam (ab111346, Abcam, Cambridge, UK). BRL37344 was from Santa Cruz Biotechnology (sc‐200154, Santa Cruz Biotechnology, Dallas, TX).

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).

Reagents were purchased or obtained from the following sources:
desamino-d-arginine vasopressin (dDAVP; desmopressin, V1005),
insulin (I1882), dexamethasone (D8893), selenium (S9133), transferrin (T1428),
triiodothyronine (T5516), and mouse epidermal growth factor (E4127) were purchased
from MilliporeSigma (Burlington, MA, USA); DMEM-F12 (31331) was from Thermo Fisher
Scientific (Waltham, MA, USA); rabbit Ab against Arg-II (sc-20151) and goat Ab
against AQP2 (sc-9882) were from Santa Cruz Biotechnology (Dallas, TX, USA); PKA
inhibitor (14-22 amide) was from MilliporeSigma; and rabbit Ab against AQP2,
Na⁺-Cl⁻ cotransporter (NCC), and
Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) were
kindly provided by Prof. Johannes Loffing (University of Zurich, Zurich, Switzerland)
(11 (link), 12 (link)). Ab against tubulin was from MilliporeSigma. Rabbit Ab against
pSer256-AQP2 (ab111346) and mouse mAb against Na⁺-K⁺-ATPase
(ab7671) were from Abcam (Cambridge, United Kingdom). IRDye 800–conjugated
affinity-purified goat anti-rabbit IgG F(c) was purchased from Bioconcept (Alschwil,
Switzerland), Alexa Fluor 680–conjugated goat anti-mouse IgG (H + L) secondary
Ab (A21057), Alexa Fluor 488–conjugated goat anti-rabbit IgG (H + L) secondary
Ab (A-11008), and Alexa Fluor 546–conjugated donkey anti-goat IgG (H + L)
secondary Ab (A-11056) were from Thermo Fisher Scientific.