Na k atpase

After the indicated transfection, HEK293 cells were rapidly washed with ice-cold phosphate-buffered saline (PBS). The total proteins were extracted using RIPA Lysis Solution (Beyotime, China) and the protein concentration was quantified by the BCA kit (Beyotime, China). A total of 30 μg of total protein was fractionated in 8% polyacrylamide gel and then transferred to nitrocellulose membranes. Membranes were then blocked with tris buffered saline (TBS) with Tween-20 (TBST) containing 5% skim milk at room temperature for 1 h to block non-specific binding sites. The primary antibody against Flag (1: 3,000, Sigma Aldrich, St. Louis, MO, USA), GAPDH (1: 3,000, ProTech, Chicago, IL, USA), or Na⁺/K⁺-ATPase (1:1,000, Abcam, Cambridge, MA, USA) was incubated overnight at 4°C and then washed with TBST. The horseradish peroxidase (HRP)-labeled secondary antibody incubation was then performed at room temperature for 1 h. Antibody binding was determined by chemiluminescence reaction. Target bands were quantified using Image J software (NIH, Bethesda, MD, USA).

Cultured cells were first washed with PBS containing Ca and Mg. Afterwards, cells were fixed using Cytofix (BD Biosciences, Franklin Lakes, NJ, USA). Permeabilization was performed using 10% donkey serum (Millipore, Burlington, MA, USA) diluted in Perm/Wash buffer (BD). Primary antibody incubation with NPHS2, SLC22A2, LRP2, Na/K-ATPase (all obtained from Abcam, Cambridge, UK), PAX2 (Life Technologies, Carlsbad, CA, USA), LHX1, AQP1, AQP2 and SLC12A3 (all obtained from Novus Biologicals, Centennial, CO, USA) occurred overnight at 4 degrees (antibody details described in Table S1). After washing three times with Perm/Wash, incubation with fluorescent-labeled secondary antibodies (Thermo Fisher Scientific) was performed in the dark at room temperature. Cells were washed again three times before the final staining of the nuclei using 4′,6-diamidin-2-phenylindol (DAPI, Sigma-Aldrich). Afterwards, DAPI solution was replaced finally with PBS with Ca and Mg. Control samples subjected to the staining procedure, including secondary antibody incubation but excluding primary antibody incubation, were used to assess the specificity of primary antibodies and for background subtraction. Cell images were obtained using the Opera Phenix High Content Screening device (PerkinElmer, Waltham, MA, USA) and final analysis was performed using Columbus Software (version 2.9, PerkinElmer).