St506 2

GST, GST-MoAtf1, GST-MoAtf1^S124D, and His-MoTup1 were expressed in Escherichia coli BL21-CodonPlus (DE3) cells. Cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF [Beyotime Biotechnology, ST506-2]) with a sonicator (Branson). Samples were centrifuged (13,000 g, 10 min) and the supernatants were transferred to a new 1.5 ml tube and stored at −70°C. The GST, GST-MoAtf1, and GST-MoAtf1^S124D supernatants were then mixed with 30 μl glutathione sepharose beads (GE Healthcare, 10265165) and incubated at 4°C for 2 hr. The recombinant GST, GST-MoAtf1 or GST-MoAtf1^S124D-bound to glutathione sepharose beads were incubated with E. coli cell lysate containing His-MoTup1 at 4°C for another 4 hr. Finally, the beads were washed with buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF, 1% Triton X-100) five times and eluted from the beads. Eluted proteins were then analyzed by immunoblot (IB) with monoclonal anti-His and monoclonal anti-GST antibodies (Li et al., 2017a (link)), respectively.

GST, GST-MoCka1, GST-MoCkb1, GST-MoCkb2 and His-MoRgs1 were expressed in Escherichia coli BL21-CodonPlus (DE3) cells (Sigma, CMC0014). Cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF [Beyotime Biotechnology, ST506-2]) with a sonicator (Branson). Samples were centrifuged (13,000 g, 10 min) and the supernatants were transferred to a new 1.5 ml tube and stored at 70°C. The GST, GST-MoCka1, GST-MoCkb1 and GST-MoCkb2 supernatants were then mixed with 30 ml glutathione sepharose beads (GE Healthcare, 10265165) and incubated at 4°C for 2 h. The recombinant GST, GST-MoCka1, GST-MoCkb1, and GST-MoCkb2-bound to glutathione sepharose beads were incubated with E. coli cell lysate containing His-MoRgs1 at 4°C for another 4 h. Finally, the beads were washed with buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF, 1% Triton X-100) five times and eluted from the beads. Eluted proteins were then analyzed by immunoblot (IB) with monoclonal anti-His and monoclonal anti-GST antibodies, respectively.