Immobilized template assays were performed as described (Chen et al. 2009 (link)). A mouse Ucp1 enhancer fragment was prepared by PCR using biotinylated oligonucleotides and immobilized on streptavidin-conjugated magnetic beads (Dynabeads M280 streptavidin, Invitrogen). After incubating the beads in blocking buffer (50 mM Tris-HCl at pH 7.5, 100 mM KCl, 0.01% NP-40, 1 mg/mL BSA, 0.5 mM PMSF, 10 mM DDT, 10 μg/mL salmon sperm DNA [GE Healthcare], 10 μg/mL poly dI–dC [Roche]), purified proteins were added to the reaction and incubated for 30 min at room temperature. The beads were then washed with wash buffer (50 mM Tris-HCl at pH 7.5, 100 mM KCl, 0.01% NP-40, 0.5 mM PMSF, 0.5 mM DTT) three times, and the bound proteins were eluted by boiling in 1× Laemmli sample buffer and analyzed by immunoblot. The standard 100-μL reaction contained 5 μL of beads, 400 ng of DNA fragment, 40 ng of TRα, 40 ng of RXRα, 40 ng of PPARγ, 400 ng of MED1, 400 ng of PGC-1α, and 400 ng of PRDM16.