Glutathione sepharose beads

Due to the difficulty of expressing and purifying intact PuM90, the CDSs of the PuM90 RNA binding domain (PuM90-RBD) and the PuM90 RNA binding domain with three amino acid residues mutated in all eight repeats (PuM90-RBD^AAA) were separately inserted into the pGEX-4T-2 vector (containing the glutathione S-transferase [GST] tag; GE Healthcare Life Science) for in vitro assays. The plasmids (GST empty vector, GST-PuM90-RBD, and GST-PuM90-RBD^AAA) were transformed into E. coli strain BL21 (DE3). A 500-mL culture of E. coli BL21(DE3) cells was grown at 37°C to an optical density at 600 nm of 0.5, after which gene expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (Sigma) for 4 h at 28°C. After lysing of cells, the recombinant proteins were purified on glutathione Sepharose beads (Sangon Biotech, Shanghai, China) and eluted with 5 mM glutathione dissolved in Tris-buffered saline. The concentration of purified proteins was determined using a bicinchoninic acid protein assay kit (Sangon Biotech). Protein purity was assessed via SDS-PAGE.

The coding sequence of ARFTF17 was amplified using primers ARFTF17-PET30F and ARFTF17-PET30R and cloned into the pET30a vector to create a fusion protein with a His tag. The coding sequence of MYB40 was amplified using primers MYB40-pCOLDF and MYB40-pCOLDR, and cloned into the pCold vector to create a fusion protein with the GST tag at NH-terminus. Recombinant proteins were produced in Escherichia coli BL21 (DE3) (EC1002) induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 20 °C for 20 h. The proteins were purified on Ni-NTA agarose (QIAGEN, 30210), following the manufacturer’s instructions. For pull-down assays, equal amounts of bait and prey proteins were incubated at 4 °C overnight in binding buffer (20 mM Tris-HCl, pH 8.0, 150 mM of NaCl, 0.2% [v/v] Triton X-100, 10% (v/v) glycerol, EDTA free protease inhibitor1025 cocktail (Roche, 4693132001). The protein complexes were recovered with glutathione Sepharose beads (Sangon Biotech, C650031), which were washed three times with buffer (50 mM Tris-HCl, pH 8.0, 140 mM of NaCl, 0.1% [v/v] Triton X-100, and EDTA free protease inhibitor1025 cocktail (Roche, 4693132001). After removing unbound proteins, those bound were analyzed by immunobloting using anti-GST (Abmart, M20007) and anti-ARFTF17 antibodies. The primers used in this assay are listed in Supplementary Table 1.