Escherichia coli bl21 cells

GST or GST-tagged plasmids were transformed into Escherichia coli BL21 cells (TianGen, China) and induced with 0.1 mM IPTG (Sigma-Aldrich, USA) overnight at 28 °C and then purified using glutathione–Sepharose 4B beads (GE Healthcare, USA). HIS-tagged plasmids were transformed and induced in the same way, but purified using HIS agarose beads. Equal amounts of individual HIS-fusion protein were incubated with GST-fusion proteins (from E. coli) in TEN buffer (10 mM Tris·HCl pH 8.0, 1 mM EDTA, 100 mM NaCl) for 4 h at 4 °C. The samples were then washed three times in TEN buffer by centrifugation at 94 × g at 4 °C for 1 min and the precipitated components were analyzed by immunoblotting.

For pull-down assays, the full-length coding region of M. mediator VRF1 without signal peptide was cloned into pGEX-4T-1vector, and the full-length coding sequence of H. armigera Dorsal was cloned into pMAL-c5x vector. The tagged proteins were expressed in Escherichia coli BL21 cells (Tiangen Biotech). GST-tagged (28 kDa) and GST-VRF1 fusion proteins were respectively bound to glutathione sepharose 4B beads (GE Healthcare). Then, the coupled beads were incubated with MBP-Dorsal fusion protein for 2 h at 4°C. The bound molecules were washed three times at 4°C with PBS buffer. After removing PBS, target proteins were collected with elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0). The eluted fractions were subjected to SDS-PAGE and followed by immunoblot analysis using anti-GST and anti-MBP monoclonal antibodies (1:5,000; Cwbiotech).