Anti wnk4

Kidney cortex homogenates or total cell membrane preparations were solubilized in Laemmli sample buffer, fractionated on SDS–PAGE (30 μg/well) and transferred to a nitrocellulose membrane (Thermo Scientific). Immunoblots were incubated overnight at 4°C with primary antibodies including polyclonal rabbit anti-NCC (1:3,000, Millipore), rabbit anti-phospho-NCC Ser71 (pNCC S71; 1:1,000), anti-phospho-NCC Ser91 (pNCC S91; 1:1,000), anti-phospho-NCC Thr 55 (pNCC T55; 1:1,000) (generous gifts of Dario R. Alessi, University of Dundee, Dundee, UK), anti-α-ENaC (Novus Biologicals, 1:1,000), anti-β-ENaC and anti-γ-ENaC (Antikoerperonline.com, 1:1,500), anti-WNK4 (1:2,000, Novus Biologicals), and monoclonal mouse anti-β-actin (1:5,000, Sigma) in 2% (w/v) bovine serum albumin (BSA, Sigma) in a TBS-T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Life Sciences). Specific signal was visualized by ECL kit (Amersham Life Sciences). The protein bands were quantified by Image Quant 5.0 software (Molecular Dynamics). The expression levels were normalized to Ponceau S stain.

Kidney cortex homogenate protein samples (1 mg) were incubated with 2 μg of anti-WNK4 (Novus Biologicals), anti-phosphoserine (Alpha Diagnostics), or anti-NCC (Millipore) antibody at 4°C overnight. The immune complexes were captured by adding 50 μl Protein A or G agarose/sepharose beads (Santa Cruz Biotechnology) and overnight incubation at 4°C with gentle rocking. The immunoprecipitates were collected by centrifugation at 1,000 × g for 5 min at 4°C and washed for four times in PBS, each time repeating the centrifugation step. After the final wash, the pellets were suspended in 40 μl of electrophoresis sample buffer and boiled for 2–3 min. Western blot analysis was performed as described above using a primary anti-NCC, anti-phosphoserine, or anti-WNK4 antibody.