Ae003

MBP-BES1 was used in our previous study (Zhang et al., 2014) and purified using amylose resin (NEB, Ipswich, MA, USA). UBP12 and UBP13 were cloned into the pET-28a (+) vector (His tag) and purified using NiNTA agarose (Invitrogen, Carlsbad, California, USA). Purified MBP, MBP-BES1 were incubated with equal amounts of His-UBP12 or His-UBP13 beads in His pull-down binding buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% Triton) at 4°C for 2 h. After washing 7 times with His pull-down washing buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton), the beads were collected, boiled in 50 μL 2 × SDS loading buffer for 8 min at 100°C, and the sample examined by immunoblotting using anti-His (1:5,000 dilution, ABclonal, China, AE003) and anti-MBP (1:5,000 dilution, ABclonal, AE016) antibodies. The antibodies source details are listed in Supplemental Table S2. The primers used for plasmid construction are listed in Supplemental Table S1.

All proteins used in this study were expressed in Escherichia coli BL21 (induced with 0.5 mm IPTG at 16 °C overnight) and purified using glutathione beads (Smart-Lifesciences, SA008100) or Ni-IDA Beads (Smart-Lifesciences, SA003025). The GST or GST-WEE1 proteins coupled to glutathione beads were incubated with GCN20-HIS in binding buffer (140 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 , and 1.8 mm KH 2 PO 4 , pH 7.4) for 2 h at 4 °C. The beads were washed 3 times with washing buffer (140 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 , 1.8 mm KH 2 PO 4 , and 1% Triton X-100, pH 7.4). The proteins were eluted by incubating the beads in 2 × SDS loading buffer at 100 °C for 8 min. Both the input and pull-down samples were subjected to immunoblotting using anti-GST (1:4,000, Abclonal, AE001) or anti-HIS (1:4,000, Abclonal, AE003) antibodies.

The HIS-GCN20 proteins were incubated with GST-WEE1 or GST-WEE1 kd in phosphorylation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm MgCl 2 , 0.1% Nonidet P-40, 0.1% Triton X-100, 500 µm ATP, and 1 mm PMSF) at 30 °C for 1 h. The reactions were stopped by adding 5 × SDS loading buffer and subjected to SDS-PAGE using gels with or without 25 μm Phos-Tag (Wako, 300-93523). The proteins were subjected to immunoblotting using anti-GST (1:3,000, Abclonal, AE001) and anti-HIS (1:3,000, Abclonal, AE003) antibodies.

In vitro pull-down assays were performed as described previously (Zhang et al. 2021) . Full-length HLS1 and SIZ1 were cloned into pMAL-c2x (MBP tag) and pET-28a (+) vector (His tag), respectively (Zhang et al. 2021) . MBP-HLS1 was purified using amylose resin (NEB), and His-SIZ1 was purified using NiNTA agarose (Invitrogen). Purified MBP or MBP-HLS1 beads were incubated with equal amounts of His-SIZ1 in MBP pulldown binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA) at 4 °C for 2 h. After washing 7 times with MBP pull-down washing buffer [200 mM NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet P-40, 20 mM Tris-HCl, pH 8.0], the beads were collected, boiled in 50 μL 2× SDS loading buffer for 8 min at 100 °C, and the sample examined by immunoblotting using anti-His (1:5,000 dilution, ABclonal, China, AE003) and anti-MBP (1:5,000 dilution, ABclonal, China, AE016) antibodies. The primers used for plasmid construction are listed in Supplemental Data Set 1.