Western Blot
HFD-mice were orally administered with vehicle or HSG4112 for 11 days and were sacrificed for extraction of hypothalamus and interscapular adipose tissues. Western blotting protocol was performed as previously described (Kim et al. , 2006). Briefly, a lysis buffer (20mM Tris- HCL pH 7.5, 150mM NaCl, 1mM EDTA, 2% SDS, 0.1mM phenylmethylsulfinyl fluoride, 2µg/ml leupeptin) was used. Tissues were added and dissolved, then dissolved for 1 hour at 4℃, and centrifuged (4℃, 14,000rpm) for protein extraction. The extracted protein was quantified with the Bio-RAD kit (Bio-Rad, Richmond, CA, USA) using the Bradford method(Kruger, 1994), mixed with a sample buffer containing SDS and β-mercaptoethanol, and boiled for 5 minutes at 95℃. Then electrophoresis was conducted in a 12% sodium dodesyl sulfate-polyacrylamide gel and the protein was transferred to a nitrocellulose membrane, reacted in each primary antibody (phospho AMPK alpha 1 (T183) and 2 (T172), and UCP1 from Abcam, Cambridge, United Kingdom) for 3 hours at room temperature, washed, reacted for 1 hour in the secondary antibody, washed, reacted in an ECL reagent (Santa Cruz, USA). Finally, autoradiography was conducted, and intensity was calculated using ImageJ software.