Anti α enac

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.

For immunohistochemistry, 5-μm-thick paraffin sections of paraformaldehyde (PFA)-fixed kidneys were prepared. Before immunofluorescence staining, dewaxed sections were pretreated with blocking solution containing 5% normal goat serum in PBS with 0.1% bovine serum albumin and 0.3% Triton X-100 for 60 min. Without rinsing, sections were incubated with polyclonal rabbit anti-NCC (Millipore, 1:500) or anti-α-ENaC (Novus Biologicals, 1:500) antibodies at 4°C overnight. After washing, sections were incubated for 1.5 h with goat anti-rabbit Alexa 488 or goat anti-rabbit Alexa 568 secondary antibodies (Invitrogen, 1:400), respectively. Controls were performed by omitting primary antibodies. The slides were analyzed on a Zeiss LSM 510 Axioplan 2 confocal microscope equipped with a 63 × oil immersion lens (NA 1.3). Individual fluorochromes were simultaneously excited by lasers at 488- and 543-nm wavelengths with appropriate filter sets for the emitted light to avoid crosstalk. Images were merged using Adobe Photoshop.