Emsa gel shift kit

The CYP6B6 HE1 element was prepared as the DNA probe for the electrophoretic mobility shift assay (EMSA). The negative control contained only HE1 probe without HaADH5 protein. For specific competition, excess unlabeled HE1 DNA was added. For non‐specific competition, a 300 bp non‐correlated sequence was added and denoted as bHLH that was a basic helix‐loop‐helix gene from Chenopodium glaucum. The EMSA/Gel‐shift kit (Beyotime) was used for the reaction. Protein‐bound probes were separated from free probes on 6% (w/v) non‐denaturing PAGE in Tris‐borate ethylenediaminetetraacetic acid buffer. All DNA bands were transferred onto Amersham Hybond‐N⁺ Membrane (GE Healthcare, Waukesha, WI, USA) using the electrode diverting method. DNA probe labeling and subsequent color detection were performed using the DIG High Prime DNA Labeling and Detection Starter Kit ΙΙ (Roche, Basel, Switzerland) according to the manufacturer's protocol. Finally, the membrane was detected using an ImageQuant LAS4000 imager.

Nuclear extracts were prepared by using Nuclear Protein Extraction Kit (Beyotime, Shanghai, China). Two sets of double-strand oligonucleotides with or without biotin-label centered on the rs71630754 were synthesized by TaKaRa Bio (Kyoto, Japan). EMSA/Gel-Shift Kit (Beyotime) was used to detect the DNA binding ability. Nuclear extracts were incubated for 20 min with unlabeled probes at a gradient concentration. For the super-shift assay, we first used JASPAR (https://jaspar.genereg.net/) to predict the possible binding transcription factors of the rs71630754. And gradient concentration of transcription factor 3 (TCF3) antibody (ab228699, Abcam, Cambridge, MA, USA) was incubated together with the nuclear extract and labeled probes for 20 min. After that we added the labeled oligonucleotide probes at room temperature, those reaction mixtures were loaded on an 8% polyacrylamide electrophoresis gel. The result was detected by SuperSignal West Femto Trial Kit (Thermo, Rockford, IL, USA).