2.6. PPARγ-Dependent Transactivation Assay

PPARγ-dependent transactivation assays were performed after cotransfection of Clover-hPPARγ encoding vectors together with the Firefly and Renilla luciferase genes containing vectors p(AOX)₃-TK-Luc [27, 28] and pRL-CMV (Promega GmbH) as previously described [23]. In parallel to the Clover-hPPARγ wild type construct, also cysteine to alanine mutants regarding hPPARγ (C109A, C112A, C126A, C129A, C146A, C150A, C160A, C163A, C168A, and C284A) was used. Transactivation was measured using a 96-well plate format in a Mithras LB940 multimode reader (Berthold Technologies, Bad Wildbad, Germany). Relative luminescence units (RLU) were calculated in a dual luciferase approach, where Firefly fluorescence was normalized to Renilla. Transfection efficiencies of all vectors (PPARγ wt and mutants) were normalized to Renilla fluorescence as well.