Kidney Lysates Preparation and Western Blot Analyses

Frozen tissues were homogenized using buffer containing Tris/HCl 50 mM at pH 7.5, EDTA 1mM, EGTA 1 mM, sucrose 0.27 M, NaF 50 mM, and Na-pyrophosphate 5 mM in addition to protease inhibitors purchased from Roche (Complete, no. 11836145001). Protein homogenates were then centrifuged at 10,000×g for 10 minutes at 4°C, supernatants were collected, and protein concentration was measured using the BradFord method (no. UPF86420; Uptima). For ROMK detection, an additional ultracentrifugation step at 100,000×g for 1 hour was performed for membrane enrichment. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes using a wet transfer apparatus (Bio-Rad). Membranes were blocked with 5% nonfat milk in TBS and 0.1% Tween 20 for 40 minutes and incubated with primary antibodies overnight at 4°C. Secondary antibodies were applied for 2 hours at room temperature. A list of the antibodies used in the study is available in Supplemental Table 2. Western blot data were quantified using ImageJ software.