Co-IP Assay

For the Co-IP assay, the full CDS of OsABCI7 was cloned into pYBA1132-GFP, and the CDS of OsHCF222 was cloned into pYBA1132-HA, respectively. The constructs were transiently expressed in N. benthamiana leaves via A. tumefaciens-mediated transfection. About 60 h after transfection, fresh N. benthamiana leaves were harvested for total protein extraction using the buffer (0.4 mm Tris-HCl, pH 7.5, 5 mm NaCl, 6.25 µm MgCl₂, 10 µm EDTA, and 1% Triton X-100) with proteinase inhibitor cocktail (Roche, 04693159001). The supernatant of total proteins was collected after centrifugation at 12,000g for 20 min at 4°C. The Pierce HA-tag magnetic IP/co-IP kit (88838X) was used for the Co-IP assay following the manufacturer’s manual. Anti-GFP (Abmart, M200045) and anti-HA (CWBIO, CW0092M) antibodies were used for detection in immunoblot analysis.