Production and Purification of Recombinant IbNAC43

The full-length IbNAC43 cDNA was amplified with specific primers and fused into the BamHI and EcoRI sites of the expression vector pET-28a (Novagen; https://www.merckmillipore.com/CN/zh). The construct vector was introduced into Escherichia coli BL21 (DE3) cells to produce recombinant 6×His-IbNAC43 proteins induced by 0.03 mM isopropylthio-β-galactoside (IPTG) and then grown at 16°C. The 6×His-IbNAC43 protein was purified with Ni-NTA Agarose (Forscience, Beijing, China) as described previously (Zhang et al., 2022 (link)). Labeled probes with biotin at their 5′-ends were used as binding probes, while unlabeled probes were used as competitors. The primer and probe sequences are listed in Supplementary Table S1. The electrophoretic mobility shift assay (EMSA) was performed using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions.