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.