Srebp 1

On day 2, the differentiated adipocyte cells were cultured in the presence or absence of EPK (25 or 50 μg/mL) or LA (200 μM) for 6 days. Cell were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 % NP-40, 0.25 % deoxycholic acid-Na, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF, and protease-inhibitor cocktail). The proteins in the samples were quantified using Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA), separated by electrophoresis on 8 to 10 % SDS-PAGE gels, and electrotransferred onto a Hybond ECL transfer membrane (Amersham Pharmacia, Piscataway, NJ). The membranes were blocked in 3 % nonfat skim milk and probed with primary antibodies for PPARγ (Novus, Littleton, CO, USA), C/EBPα (Cell Signaling Tech., Danvers, MA, USA), p-AMPK (Cell Signaling Tech., MA, USA), AMPK (Cell Signaling Tech., MA, USA), adiponectin (Cell Signaling Tech., MA, USA), FABP (Cell Signaling Tech., Danvers, MA, USA), HMGCR (Bioss Antibodies, MA, USA), SREBP-1 (Sigma, St. Louis, MO, USA), or β-actin (Sigma, St. Louis, MO, USA) overnight. Subsequently, they were exposed to horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies. Protein expression was examined by using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ).