Fig. 5.

An EMSA validated the PeWRKY1 interaction with the W-box in the PeHA1 promoter. The purified PeWRKY1–GST protein obtained by prokaryotic expression was used for the in vitro assay, and GST protein was used as a negative control. The purified proteins were combined with biotinylated W-box-containing dsDNA probe (1×), and unlabeled probes (Competitor) were subjected to cold competition experiments. The biotin-unlabeled mutant probes (Mut, TTGACC mutated into TTAGCC) were used to confirm the binding specificity of the W-box to PeWRKY1. ‘+’ and ‘–’ indicate the presence or absence, respectively, of proteins and probes in the loading mixture. The cold probe concentrations are 5× and 10×, and that of the Mut probe is 5×. The concentration of the polyacrylamide gel was 4%.