ABCC11 Protein Expression Analysis

The expression of ABCC11 protein in plasma membrane vesicles was examined by immunoblotting, as described previously [4 (link),9 (link)] with minor modifications. Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond^® ECL^TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na⁺/K⁺-ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively. All antibodies were used in TBST-skim milk. HRP-dependent luminescence was developed using the ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging Analyzer Fusion Solo 4^TM system (Vilber Lourmat, Eberhardzell, Germany).