Western blotting was performed as previously described [50 (link)]. The protein concentration was measured using the DC protein assay (Bio-Rad, Hercules, CA, USA). The primary antibodies included anti-Ucp1 (Sigma-Aldrich, St. Louis, MO, USA), anti-COXIV, anti-phospho-p44/42 MAPK (ERK1/2), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-Pparγ, and anti-β-actin (all purchased from Cell Signaling Technology, Danvers, MA, USA). The secondary antibody staining was visualized using a chemiluminescent horseradish peroxidase substrate (Millipore, Burlington, MA USA).
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