*4.6. Western Blot Analysis*

The differentiated 3T3-L1 cells were harvested using a cell scraper, lysed with radioimmunoprecipitation assay buffer containing protease inhibitor cocktail at 4 ◦C for 1 h. The protein concentration was determined using Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of extracted proteins were separated on 8–13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane. The each membrane was blocked 5% skim milk at room temperature for 1 h, and then incubated primary antibodies such as β-actin, C/EBPα, C/EBPβ, PPARγ, FAS, ap2, GLUT4, ATGL, phospho-HSL, HSL, phospho-AMPK, AMPK, phospho-acetyl-CoA carboxylase (ACC), and ACC at 4 ◦C for overnight. The next day, each membrane was washed with PBS-T, and then incubated with secondary antibodies at room temperature for 1 h. The protein bands were activated with enhanced chemiluminescence solution, visualized with Davinch-chemiTM Chemiluminescence Imaging System (Core Bio, Seoul, Korea), and quantified with Image-J program (National Institutes of Health, Bethesda, MD, USA).
