Ni ida beads

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.