Sab5200111

UT‐B proteins were detected using the previously characterized hUT‐Bc19 antibody (Walpole et al., 2014), which had been raised against a 19 amino acid peptide (NH₂‐EENRIFYLQAKKRMVESPL‐COOH) corresponding to the C‐terminal end of human UT‐B1. Commercially available antibodies for AQP3 (SAB5200111, Sigma‐Aldrich), NaKATP (sc‐28800, Santa Cruz Biotechnology) and GAPDH (sc‐66163, Santa Cruz Biotechnology) were also used, together with horseradish peroxidase conjugated secondary anti‐rabbit IgG antibody (65–6120, Invitrogen) or anti‐mouse IgG antibody (61–6520, Invitrogen).

Cells were lysed in RIPA buffer (supplemented with a protease and phosphatase inhibitor cocktail) and homogenates were solubilized in Laemmli buffer [61 (link)]. 15–30 μg solubilized proteins were subjected to 12.5% SDS-polyacrilamide gel electrophoresis and transferred to the Hybond-P PVDF Membrane (GE Healthcare, Chicago, IL, USA) by electroelution. The membranes were incubated overnight with anti-AQP3 antibody produced in rabbit (SAB5200111; Sigma, Italy) diluted 1:1000 in TBS and 0.1% Tween-20. The membranes were washed and incubated for 1 h with goat anti-rabbit IgG antibody, peroxidase conjugated (AP132P; Millipore, Burlington, MA, USA) diluted 1:100,000 in blocking solution. The bands were detected with ECL™ Select western blotting detection system (GE Healthcare). Pre-stained molecular weight markers (ab116028, Abcam) were used to estimate the molecular weight of the bands. Blots were stripped and re-probed with RabMAb anti β-2-microglobulin antibody ([EP2978Y] ab75853; Abcam, 1:10000) or with anti β-actin polyclonal antibody (Cat.n. AB-81599; Immunological sciences, USA, 1:2000) as housekeeping.

To investigate the protein localisation and abundance of UT-B2, AQP3 and MCT1, various antibodies were employed. A previously characterised polyclonal UT-B antibody, UT-Bc19, was raised in rabbit against an immunizing peptide corresponding to the last 19 amino acids of human UT-B1 [23 (link)]. For AQP3, a commercial polyclonal antibody AQP3-SAB (SAB5200111, Sigma-Aldrich, Arklow, Ireland) was used, which was raised in rabbit against the C-terminus of rat AQP3 (undisclosed sequence). Another commercial polyclonal antibody raised in chickens against the C-terminus of rat MCT1 was also utilised (AB1286-I, Millipore, Livingston, UK). Finally, for Na/K ATPase, a mouse-derived monoclonal antibody was used (sc-48345, Santa Cruz Biotechnology, Dallas, TX, USA). The corresponding HRP-linked secondary antibodies used were also used for this study: anti-rabbit (656120, Biosciences Ltd., Cambridge, UK); anti-chicken (A16054, Biosciences Ltd., Cambridge, UK); anti-mouse (32430, Biosciences Ltd., Cambridge, UK).