Site-Directed Mutagenesis of Transcription Factor Binding Sites

SDM was performed with QuikChange^TM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518), as described in the company’s protocol. Briefly, amino acid exchanges at the Klf9 sites (RKBE 1 and RKBE 3) mutants (RKBE 1; GC to TT and RKBE 3; AC to TT) were produced by point mutations in the human promoter of Prdx6-linked to CAT plasmid as described earlier [11 (link)]. To prepare Nrf2/ARE (ARE 3 and ARE 4) sites mutants, base A was changed to C, and base T to G at ARE sequences in the mouse Klf9 promoter-linked to CAT reporter plasmid by using site-directed mutagenesis. The following complementary primers were utilized (changed nucleotides are in red boldface type and underlined).