Na935v

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).

In human tissue culture cells, after 72 hours of siRNA knockdown, protein was harvested by scraping, rinsed in PBS, and lysed by vortexing in AZ lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1% Igepal, 0.1% SDS, 5 mM EDTA pH 8.0) containing protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche, 11697498001) for 15 minutes at 4°C. The lysate was spun at 21,000 x g for 15 minutes at 4°C. The supernatant was removed, and total protein was quantified by Bradford assay (Bio-Rad). Samples were analyzed via SDS-PAGE. Antibodies used include α-p53 (1:5,000 Santa Cruz, sc-126 HRP), α-β-actin (1: 30,000 Sigma-Aldrich A1978), α-PAX9 (1:1,000, Cell Signaling Technology D9F1N), α-vinculin (1:20,000 Abcam ab18058), and α-puromycin (1:10,000 Kerafast 3RH11). HRP conjugated secondary antibodies include α-mouse (1:10,000 GE Healthcare NXA931) and α-rat (1:10,000 GE Healthcare NA935V). Images acquired either by film developing or by digital imaging using the BioRad ChemiDoc Imaging System.
For X. tropicalis western blotting, protein was harvested according to the TRIzol (Life Technologies 5596018) protocol for 2 replicates, and as in [70 (link)] for 1 replicate. Samples were analyzed via SDS-PAGE. Antibodies included α-p53 (1:800, Thermo Fisher MA1-12549) and α-GAPDH (1:5,000 Ambion AM4300). HRP conjugated secondary antibodies include α-mouse (1:10,000 GE Healthcare NXA931).