Fluorchem instrument

Western blot analysis was performed on plasma membrane fractions prepared from different samples as described above. Equal amounts of protein (10 μg) were denatured in a sample buffer (10 mM Tris-HCl, pH 7, 12% glycerol, 2% SDS, 0.01% bromophenol blue and freshly added 1-mM dithiothreitol) and separated by electrophoresis on an 8% polyacrylamide gel. Proteins on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane that was blocked with 5% milk or BSA in TBS (20 mM Tris, pH 7.5, and 150 mM NaCl) with 0.1% Tween-20 and then incubated with primary antibody against Na-K-ATPase α1 (Cat# 05-369, Millipore sigma, St.Louis, MO, USA) or Na-K-ATPase β1 (Cat# ab2873, Abcam, Cambridge, MA, USA) overnight at 4°C. Membranes were washed three times each with TBS and TBST, followed by incubation with secondary antibody for 1 h. Membranes were washed again three times each with TBS and TBST. ECL Western blotting detection reagent (GE healthcare Bio-Sciences, Piscataway, NJ, USA) was used to detect the immobilized protein. The chemiluminescence was detected using FluorChem instrument (Alpha Innotech, San Leandro, CA, USA) and analyzed with its software. Ezrin (Cat# ab4069, Abcam, Cambridge, MA, USA) antibody was used to normalize the expression levels of proteins in plasma membrane fractions.

BBM protein extract solubilized in a RIPA lysis buffer (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) was used for the Western Blot analyses. Equal amount of proteins were denatured in sodium dodecyl sulfate sample buffer (20 mM Tris pH 7, 12% glycerol, 2% SDS, 0.01% bromophenol blue, and freshly added 1 mM Dithiothreitol) and separated by electrophoresis on a 8% Gel (Bio-Rad Laboratories, Hercules, CA). Proteins on the gel were transferred to a polyvinylidene membrane which was blocked with 5% bovine serum albumin in TBS (20 mM Tris, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and then incubated overnight at 4°C with goat polyclonal antibody against B0AT1 followed by incubation with anti-goat IgG conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA) for an hour at room temperature. The primary antibody was obtained through the custom antibody services provided by Invitrogen. ECL reagent for Western blotting detection (RapidStep™ ECL Reagent, Millipore) was used to detect the immobilized protein. The resultant chemiluminescence was detected using biomax film (Kodak, Rochester, NY) and the intensity of the bands was analyzed with FluorChem^TM instrument (Alpha Innotech, San Leandro, CA).