Total nedd4 2

For determination of protein abundance, total protein lysates from Ishikawa cells were prepared by lysing cells in RIPA buffer. Protein yield was quantified using the Bio-Rad DC protein assay kit (Bio-Rad). Equal amounts of proteins (30 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide (SDS) gel before electrotransfer onto the PVDF membrane (Amersham Biosciences, Freiburg Germany). Nonspecific binding sites were blocked by overnight incubation with 5% nonfat dry milk in Tris-buffered saline with 1% Tween (TBS-T; 130 mmol/L NaCl, 20 mmol/L Tris (pH7.6) and 1% Tween). Primary antibodies used were αENaC (1:500, #E4653,Sigma, Taufkirchen, Germany), phospho-SGK1 (1:1000, #5599, Cell Signaling, Erlangen, Germany), phospho-NEDD4-2 (1:1000 #ab168349, Abcam, Cambridge, UK), Total-NEDD4-2 (1:1000, #40135, Cell Signaling), Total-SGK1(1:1000 #3272, Cell Signaling) and β-actin (1:1000, #4967, Cell Signaling), which was used as a loading control. For detection, a secondary anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (1:2000, #7074, Cell Signaling) or secondary anti-mouse IgG antibody conjugated with HRP (1:2000, #7076, Cell Signaling) was used. Protein complexes were visualized with a chemiluminescent detection kit (Invitrogen). All experiments were performed in 3 or more cell cultures. Bands were quantified with ImageJ Software.