Pcdna3.1 his lacz

A luciferase reporter assay was performed as described previously (Kim et al., 2019 (link); Yu et al., 2016 (link)). Briefly, 293T (3.5 × 10⁵ cells/ml) or 3T3-L1 (1 × 10⁵ cells/ml) cells cultured in 24-well plates were cotransfected with reporters and an appropriate combination of epitope-tagged eukaryotic expression plasmids for TDAG51, PPARγ or RXRα in triplicate using TurboFect reagent (Fermentas, USA) according to the manufacturers’ instructions. Plasmids P_T51-3K-Luc (0.1 µg), PPARγ-Luc (0.1 µg), aP2-Luc (0.1 µg), and pcDNA3.1/His/LacZ (0.1 µg, Invitrogen) were used as reporters. The reporters were cotransfected with the epitope-tagged expression plasmids (0.1-0.5 µg). The β-galactosidase activity derived from the expression of pcDNA3.1/His/LacZ was used as an internal normalization control for transfection. For the reporter assay in 3T3-L1 cells, P_T51-3K-Luc-transfected 3T3-L1 cells were differentiated with adipocyte differentiation media for 2 days. For the treatment of rosiglitazone, the transfected cells were treated with 5 µM rosiglitazone for 36 h, as described previously (Dowell et al., 2003 (link)). At 24 h posttransfection, reporter activities were measured using a luciferase assay kit (Promega) and β-galactosidase assay kit (Applied Biosystems, USA) according to the manufacturers’ instructions.