Ni nta his bind resin

The CDS of CsESE3 was merged into the pETDuet vector to generate pETDuet- CsESE3 vector with an N-terminal His-Tag. The construct was then introduced into the Escherichia coli strain BL21 and Rosetta (DE3), and protein expression was induced by 1 mM IPTG. The CsESE3 protein was purified using Ni–NTA His Bind Resin following the manufacturer’s instructions (Sangon Biotech, China). About 33-bp promoter fragments containing the predicted binding sites from gene CsPLIP1 and CsPLIP_Like were 5’ FAM-labeled (GenScript, China). 100 μM of unlabeled probes (the same or mutated oligonucleotides) were excessively supplied as cold competitors or mutated competitors. Detailed oligonucleotide information is listed in Table S4. The binding reaction of CsESE3 protein and probes was performed according to the method of Gong et al. (2021b (link)). Cold competitor probes were added at a maximum of 28-fold excess.

Cells were collected with lysis buffer ((50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, and 1 mg/mL each of leupeptin, pepstatin, and aprotinin). One milligram of proteins was then immunoprecipitated overnight at 4 °C with the indicated antibody or Ni-NTA His Bind Resin (Sangon Biotech, Shanghai, China). After extensive washing with lysis buffer, the immunocomplexes were analyzed by western blotting assay. For ubiquitination assays, HEK293T cells or U2OS cells were cotransfected with plasmids encoding HA-Ub, His-tagged DYRK1A, and the indicated TP53, Myc3-MDM2 WT or MDM2 C464A, siRNA duplexes specific to MDM2 or negative oligo. Followed by treated with MG132, the cell lysates were prepared, and DYRK1A or p53 precipitates were isolated with Ni-NTA or protein A/G-agarose, and then subjected to western blotting.

The induced cells were harvested at 5,000 rpm at 4°C and washed twice with Tris-HCL buffer (pH 7.8, 50 mM). Then, the cells were disrupted in a constant system cell breaker (Constant Systems) using a pressure setting of 30 kpsi. The supernatant (crude enzyme) was collected by centrifugation (Hitachi) at 12,000 rpm for 20 min at 4°C and purified by Ni-NTA His Bind Resin (Sangon Biotech). Tris-HCl (20 mM, pH 8.0) containing 30 mM imidazole was set as the wash buffer, while the elution buffer contained 250 mM imidazole. Then the eluate was concentrated using the tubular ultrafiltration modules (Millipore Merck, 10 kDa) at 4,500 rpm and repeatedly washed with Tris-HCl buffer to remove imidazole. The protein concentration was determined by the Bradford Protein Assay Kit.