Phosphatase inhibitory cocktails 2 and 3

Liver tissue homogenates were prepared by homogenizing the tissue in lysis buffer (20 mM Tris, pH 7.4; 150 mM NaCl; and 0.5% Triton X-100) or RIPA buffer (150 mM NaCl; 50 mM Tris, pH 8.0; 2 mM EDTA; 1% Nonidet P-40; and 0.1% SDS) supplemented with a protease inhibitory cocktail tablet (Roche, Indianapolis, IN, USA) and phosphatase inhibitory cocktails 2 and 3 (Sigma-Aldrich). Lysates were cleared by centrifugation and analyzed by gel electrophoresis. Protein concentration was determined via Bradford protein assay (Bio-Rad, Hercules, CA, USA) using BSA as a standard, and verified by Coomassie blue gel staining. Lysates (45 μg) were resolved by SDS-PAGE (Bio-Rad) and then transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline (0.137 M NaCl, 0.025 M Tris, pH 7.4) containing 0.1% Tween-20 (T-TBS). Antibodies were diluted according to the manufacturers’ recommended protocols. Protein signals were visualized using an enhanced chemiluminescence (ECL) reagent (SuperDetect^TM ECL Western Blotting Detection Reagent, DaeMyung Science Co., Ltd, Seoul, Korea). Finally, membranes were exposed to imaging film (Kodak BioFlexEcono Scientific Supplies, Citrus Heights, CA, USA), which was developed using a Kodak X-OMAT 1000A Processor.

Liver tissue homogenates were prepared by homogenizing the tissue in RIPA buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 2 mM EDTA; 1% Nonidet P-40; 0.1% SDS) supplemented with a protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) and phosphatase inhibitory cocktails 2 and 3 (Sigma-Aldrich). Lysates were cleared by centrifugation and analyzed by gel electrophoresis. Protein concentration was determined via the Bradford protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as a standard, and verified by Coomassie blue gel staining. Lysates (40 μg) were resolved by SDS-PAGE (Bio-Rad) and then transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline (0.137 M NaCl, 0.025 M Tris, pH 7.4) containing 0.1% Tween-20. Antibodies were diluted according to the manufacturers’ recommended protocols. After incubation with an enhanced chemiluminescence (ECL) reagent (SuperDetect^TM ECL Western Blotting Detection Reagent, DaeMyung Science Co., Ltd, Seoul, Korea), the membranes were exposed to film (Amersham Hyperfilm TM ECL, GE Healthcare Limited, Buckinghamshire, UK) to detect protein signals.