Anti flag

To perform the Co‐IP assays, different combinations of proteins were co‐expressed in N. benthamiana leaves via injection, including pRI‐101‐35S::GFP and pCambia2300‐35S::MdPRP6‐Flag, pRI‐101‐35S::MdRAD23D1‐GFP and pCambia2300‐35S::MdPRP6‐Flag, pCambia2300‐35S::GFP and pRI‐101‐35S::MdRAD23D1‐mCherry, and pRI‐101‐35S::MdRAD23D1‐mCherry and pCambia2300‐35S::MdRPN13‐GFP. Total proteins were extracted using an extraction buffer containing 50 mm Tris–HCL, 150 mm NaCl, 5 mm EDTA, 1% NP‐40, 10% glycerol, 1 mm PMSF, 5 mm DTT, and 1× complete protease inhibitor cocktail. Anti‐GFP magnetic beads were used for Co‐IP assays according to the manufacturer's manuals (Beyotime, Shanghai, China). Briefly, 10 μL pre‐prepared Anti‐GFP magnetic beads were added to each 500 μL of protein sample and incubated at 4 °C overnight with gentle shaking. The beads were then washed three times with extraction buffer, and the immunoprecipitated proteins were eluted according to the protocol of Beyotime. Protein blot analysis was performed using anti‐GFP, anti‐Flag, or anti‐mCherry antibodies (Yeasen, Shanghai, China).