Lef/Tcf luciferase reporter assay

RKO cells (0.5×10⁵) were plated in 12-well dishes and incubated overnight at 37°C. The cells were transfected with hPar2-wt or hPar2 mutants (i.e. hPar2-truncated, hPar2-356 or hPar2-368) plasmids, human Lef-1 TOPflash (Tcf Optimal Promoter + luciferase, T cell factor (Tcf) reporter plasmid containing two sets (the second set in reverse orientation) of three copies of the Tcf binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame using Fugene 6 transfection reagent (Boehringer-Mannheim). CMV/β-gal plasmid was cotransfected as an internal control for transfection efficiency. After 48 h transfection, the cells were washed in PBS and luciferase assay performed with the Luciferase Reporter System (Promega, Heidelberg, Germany) according to the manufacturer's instructions, and detected on a luminometer Mithras LB940, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).