Anti gfp magnetic beads

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).

The histone acetyltransferase activity of ZmELP3 was analysed according to a previously described protocol with minor modifications (Xu et al., 2023 (link)). Vectors of Pro35S:ZmELP3A or Pro35S:ZmELP3B and Pro35S:ZmH3.2‐GFP were transformed into Agrobacterium strain GV3101, and then infiltrated into 5‐week‐old N. benthamiana leaves. After ground into powder by liquid nitrogen of the transfected tobacco leaves, the total proteins were extracted using IP buffer (10 mm Tris–HCl, pH 7.5, 0.5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P‐40 and 1% protease inhibitor cocktail). After removing cellular debris, the supernatant was added to anti‐GFP magnetic beads (Beyotime) and incubated for 3 h at 4 °C. The beads were then washed five times with IP buffer. The extracted proteins were analysed using Western blotting and detected with anti‐H3 (Abcam, ab1791, 1 : 1000), anti‐ELP3 (Invitrogen, 702 669, 1 : 1000) and anti‐H3K14ac (Abcam, ab52946, 1 : 1000).