Promoter deletion and site-directed mutagenesis
The lox-1 promoter luciferase reporter plasmid p-2336/+36-Luc, also termed pLOX-1-Luc, was used as a PCR template to generate constructs with various lengths of the gene promoter. Primers in Table 2 were used for generating the plasmids by PCR. The primers were tailed with KpnI site (forward primers) or BglII site (reverse primer), and the PCR products were subcloned into pGL3-Basic (Promega) at KpnI/BglII sites. The site-directed mutagenesis was carried out using the GeneTailor Site-Directed Mutagenesis kit (Invitrogen), according to the manufacture's instruction. The sequence of 5′-GGC ACA TTT TTT ACA AAT GTA GTG TGA CTT ACT CTC TTT GAA TTT CAG TTT C-3′ (-310 to -258) containing the putative TCF/LEF-1 binding site in the plasmid p-2336/+36-Luc, was mutated to 5′-GGC ACA TTT TTT ACA AAT GTA GTG TGA CTT ACT CTC ATA GAA TTT CAG TTT C-3′ in p-2336/+36-Mut-Luc. The primers used for mutagenesis were in Table 3. The mutations were verified by DNA sequencing.