Magnegst pull down system

The CC domain of Pikh-1 was inserted into the pF3A WG vector fused to a FLAG tag construct (Promega), while the Pikh-2_CC, and intact and truncated fragments of AvrPik-h were inserted into the pGEX-6P-1 vector to form a GST-fusion construct (GE Healthcare). The GST-fused proteins expressed in E. coli strain BL21 were used to pull down the FLAG-fused proteins synthesized in a wheat germ-derived in vitro transcription and translation system (Promega) using a MagneGST pull-down system (Promega), following the manufacturer's protocol. The proteins eluted from the beads were separated through a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). Protein blots were blocked with 3% w/v skimmed milk and then probed with either an anti-GST or an anti-FLAG monoclonal antibody (Sigma). Either goat anti-mouse (Sigma) or anti-rabbit IgG HRP conjugates (Bio-Rad) was used as the secondary antibody for subsequent detection via enhanced chemiluminescence (GE Healthcare). As an additional recognition specificity assay, two sets of the five Pik alleles, three versions of AvrPik-h alleles, as well as the Pikh-2_CC (the Pik-2 alleles share an identical CC domain) were tested by the above procedure.

GST fusion protein pulldown assays were carried out using the MagneGST pull-down system (Promega) as described essentially (33 (link), 34 (link)). Briefly, GST fusion proteins were expressed in bacteria and purified by using glutathione purification system. Purified fusion proteins were analyzed by Coomassie Brilliant blue staining following SDS-PAGE before experiments. GST fusion proteins tethered to the glutathione beads were either used immediately or stored at 4 °C for no longer than 2 days. To measure C1orf27 interaction, purified GST fusion proteins were incubated with HEK293 cell homogenates expressing GFP-C1orf27 in a total volume of 400 μl binding buffer containing 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 1% Nonidet P-40, and 10% glycerol overnight at 4 °C. After washing three times with binding buffer, the bound proteins were solubilized in SDS gel loading buffer and detected by immunoblotting using GFP antibodies.

The genuine positive was further confirmed by GST pull-down assays. The OsCYP2 and OsPEX11 were cloned into pGEX-4T-1 and pET-28a vectors, respectively, for expressing fusion protein with glutathione-S-transferase (GST) and histidine (His), in Escherichia coli strain BL21. The MagneHis Protein Purification System (Promega, V8500) and MagneGST Pull Down System (Promega, V8870) were used for fused protein purification and GST pull-down, respectively. The purified GST, GST-OsCYP2 and His-OsPEX11 proteins were analyzed with 12% SDS-PAGE and stained by coomassie brilliant blue R-250. Western blotting signals were detected by Horseradish Peroxidase (HRP) DAB (3, 3-diaminobenzidine) staining with either the His tag antibody (Genescript, A00612) or GST tag antibody (Genescript, A00130).

A GST pull‐down assay was performed using a Magne GST pull‐down system (Promega) according to the manufacturer's instructions. Briefly, proteins were expressed in Escherichia coli BL21 (DE3) overnight at 18°C, and purification of GST fusion proteins was performed with GST‐tag purification resin (P2253; Beyotime). Total cell lysates were subjected to GST pull‐down by magnetic beads overnight at 4 °C (P2138; Beyotime) with immobilized GST‐control or GST‐tagged proteins as indicated. The beads were then washed three times with chilled lysis buffer. Eluted samples and inputs were subjected to western blotting.