Anti nkcc2

DMEM-F12, newborn bovine serum, HEPES and penicillin/streptomycin were purchased from Wisent Corporation (Wisent, Canada). Hank's balanced salt solution (HBSS), nonessential amino acids, sodium pyruvate, insulin-transferrin-selenium and L-glutamine were purchased from Invitrogen Life Technologies (Paisley, Scotland). Stainless steel sieves were obtained from Merck Eurolab (Leuven, Belgium). Anti-NKCC2 (Stressmarq Biosciences Inc., Canada), anti-NLRP3 (Abcam, Cambridge, MA), anti-mPGES1 and anti-COX2 (Cayman, Ann Arbor, MI) primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

The whole kidney was lysed, and the protein concentration was determined using the Coomassie reagent. Protein (60 μg) from whole kidney lysates was denatured in boiling water for 10 min, separated via SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for 1 h with rabbit anti-NHE3 (Abcam, Cambridge, MA), anti-α-Na-K-ATPase (Abcam), anti-NKCC2 (Stressmarq Biosciences Inc., Canada), anti-ENaCα (Stressmarq Biosciences Inc., Canada), anti-ENaCβ (Stressmarq Biosciences Inc., Canada) or anti-ENaCγ (Stressmarq Biosciences Inc., Canada) at a dilution of 1:1,000 After being washed with TBS, blots were incubated with a goat anti-horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution) and visualized using ECL kits (Amersham, Piscataway, NJ USA).