Ni nta column

The full length of the open reading frame (ORF) of serpin-4 cDNA sequence (313-1,551 bp), serpin-5 cDNA sequence (166-1,116 bp) and the previously reported serpin-2 (1,185 bp) gene in Genebank (gi|117970183|) were amplified with primers PxSp4OrF, PxSp4OrR, PxSp5OrF, PxSp5OrR, PxSp2OrF and PxSp2OrR (Table 1), among which the forward and reverse primers contain restriction sites Bam HI and Not I respectively. The amplification conditions of the first PCR run were denaturing at 94°C for 5 min, then 35 cycles of 94°C for 30 s, annealing at 46°C for 30 s, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were digested with Bam HI and Not I (Thermo Scientific, USA) subcloned into pET-32a (+) vector. This plasmid construction was used for protein expression in E. coli (BL21) competent cells. The E. coli (BL21) was disrupted by an ultrasonic wave and solubilized in equilibrium buffer: 8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris base, pH 8.0, then purified with the Ni-NTA column (TransGen Biotech, China). Purified recombinant serpin proteins were used to immunize rabbits using a previously described method [65] (link).

Electrophoretic mobility shift assay was performed as described previously (Zheng et al., 2021 (link)). The full-length cDNA of PuCRZ1 was cloned into pET32a (+) vector (Invitrogen, CA, USA), and then transformed into the Escherichia coli strain BL21 (DE3) competent cell (TransGen Biotech, Beijing, China). An empty pET32a (+) vector was used as a negative control. Prokaryotic expression was performed at 16°C for 16 h with 0.5 mM isopropyl-β-d-thiogalactoside (IPTG), and then recombinant protein was purified using Ni-NTA column (TransGen Biotech, Beijing, China), and the SDS-PAGE result of purified PuCRZ1 protein in Supplementary Figure 5. Primers and probes are listed in Supplementary Table 2. Unlabeled probes were subjected to cold competition experiments. EMSA were performed using the Chemiluminescent EMSA kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The binding reactions were performed using 1 μg of 6 × His-PuCRZ1 incubated with 7.5 nM probe in binding buffer for 30 min at room temperature. The 6 × His protein was used as a negative control. The reaction mixtures were separated in 6% native polyacrylamide gel electrophoresis (PAGE) gel. Biotin activity was detected according to the manufacturer’s instructions. The EMSAs were repeated three times, and representative results are shown.

The 3-keto-DON (purity ≥ 98%) was from TripleBond (Guelph, ON, Canada). The standards were made with 5 mg/mL stock solution in acetonitrile solvent. Restriction enzymes and T4 DNA lignase were ordered from New England Biolabs (Beijing, China). The 2 × Taq plus PCR MasterMix, DL2000 DNA marker, DNA loading buffer, and GelRed were purchased from Aidlab Co., Ltd. (Beijing, China). The Ni-NTA column was purchased from TransGen Biotech Co., Ltd. (Shanghai, China). The other chemicals were from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).