Luciferase reporter assays

Plasmids expressing PPARγ or PRDM16 were described previously (33). The Ucp1 luciferase reporter was constructed by cloning the promoter fragment containing 3.1 kb of the 5′ flanking region of the mouse Ucp1 gene into the pGL3 basic vector (Promega) upstream of the firefly luciferase–encoding region. 293T cells were plated onto a 12-well plate and transiently transfected using the PEI method with plasmid expressing PRMT1V2 (100 ng) or empty vector (100 ng) and PPARγ (100 ng) and PGC1α (100 ng) or PRDM16 (100 ng) in cotransfection assays together with 250 ng of Ucp1 promoter luciferase reporter construct. Cells were lysed 48 hours after transfection. Luciferase activity was measured by the a luciferase assay kit (Promega) according to the manufacturer’s recommendations, using a PerkinElmer EnSpire 2300 multilabel microplate reader.