EMSA

The full-length ORF of MRP1 was cloned into the pCold-TF DNA Vector (Takara, Shiga, Japan). Recombinant His-TF-MRP1 was expressed in E. coli BL21 (DE3) cells by adding 0.1 mM IPTG when OD₆₀₀ reached 0.8. His-TF-MRP1 and His-TF (negative control) were purified with BeaverBeads IDA-Nickel (Beaver, Suzhou, China; cat. no. 70501-5) and used for EMSA. EMSA was performed as previously described with minor modifications (Li et al., 2015). Approximately 200 ng of purified His-TF-MRP1 and 5 ng 5′-biotin-labeled probes (WT probe or Mu probe harboring mutated TATCTA motif) were added to the reaction mixtures according to the standard protocol of the LightShift EMSA Optimization & Control Kit (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 20148X). Competition EMSA for the biotin-labeled probe was conducted by adding 50, 200-fold excess of unlabeled WT or Mu probes. Mutation EMSA for the biotin-labeled probe was conducted by mutating TATCTA motif. The samples were electrophoresed in a 6% native polyacrylamide gel. DNA and DNA–protein complexes were transferred to an Amersham Hybond-N+ membrane (GE Healthcare, Chicago, IL, USA; cat. no. RPN303B) and UV cross-linked. The biotin-labeled probes were detected using a Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 89880). Bands were visualized using the Tanon 5200 chemiluminescence imaging system (Tanon Science & Technology, Shanghai, China). Relevant primer sequences are given in Supplemental Data Set 6.