At the end of the infusion period, the mice were sacrificed, and kidneys from each group were homogenized in lysis buffer containing the following: 50 mmol/l Tris–HCl (pH 7.5), 1 mmol/l ethylene-bis(oxyethylenenitrilo)tetraacetic acid, 1 mmol/l ethylenediaminetetraacetic acid, 50 mmol/l sodium fluoride, 5 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1% (wt/vol) Nonidet P-40 (Sigma-Aldrich), 0.27 mol/l sucrose, 0.1% (vol/vol) 2-β-mercaptoethanol and protease inhibitors (Complete tablets; Co-Ro Roche, Sigma-Aldrich). Sixty micrograms from each homogenate were resolved into 10% SDS–PAGE and transferred for 1 h to polyvinylidene difluoride membranes. Membranes were blocked in 10% skim milk and incubated overnight with sheep antipNCC (T60) antibody, and after stripping, they were incubated with anti-NCC antibody, both produced by Dario Alessi from Phosphorylation Research Unit (Dundee, Scotland, United Kingdom) that were previously used and characterized by our group [7 (link),18 (link)] and anti β-actin (Santa Cruz Biotechnology Inc, Dallas, Texas, USA) antibodies. For densitometric analysis purpose, total NCC and pNCC were normalized with β-actin, then, total pNCC/NCC ratio was calculated.
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