Ubiquitinated NKCC2 was measured as previously described (7 (link)). Briefly, ubiquitinated proteins were isolated using a Ubiqapture-Q kit (Enzo Life Sciences, Farmingdale, NY) following the manufacturer’s instructions. To measure ubiquitinated NKCC2 and the effect of cGMP, the whole TAL sample was treated with the proteasomal inhibitor MG132 (20 µM). TALs were split and treated with vehicle or inhibitor for 10 min at 37°C. Vehicle or dybutyryl cGMP (db-cGMP) was then added to the respective samples and incubated for 50 min at 37°C. Once the treatment was finalized, samples were cooled with chilled PS. Suspensions were centrifuged at 120 g for 2 min at 4°C. The PS was discarded, and TALs were incubated with lysis buffer [containing 150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 2% Triton X-100, and 0.2% SDS and supplemented with protease inhibitors, namely, 10 µg/mL aprotinin, 5 µg/mL leupeptin, 4 mmol/L benzamidine, 5 µg/mL chymostatin, and 5 µg/mL pepstatin-A; pH 7.5 (Sigma)]. TALs were vigorously vortex three times for 3 s each. Each tube was spun at 12,000 g for 2 min at 4°C. The undissolved pellet was discarded. The protein content in each sample was measured in duplicate with a colorimetric assay using Bradford’s method (Pierce Biotechnology, Rockford, IL). The TAL lysate (150 μg protein) was incubated on a rocking platform at 4°C overnight with 40 μL of a 50% slurry containing Ubiqapture-Q beads in a final volume of 400 μL. The beads were centrifuged at 12,000 g for 2 min at 4°C. The supernatant was separated from the beads and saved for later measurement of nonubiquitinated NKCC2 and was also used as a loading control. The beads were washed twice with high-salt buffer (500 mM NaCl and 50 mM HEPES; pH 7.4) and twice with no-salt buffer (50 mM HEPES; pH 7.4). Proteins were eluted from the beads by boiling in 60 μL of SDS Laemmli loading buffer containing 50 μM dl-dithiothreitol and 5% β-mercaptoethanol. Proteins from the supernatant and proteins eluted from the beads were separated by SDS-PAGE (6% gels) and transferred to Immobilon PVDF membranes (Millipore, Bedford, MA). NKCC2 was detected by Western blot analysis.
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