In Vitro Pull-Down Assay

GST or GST-AtMMS21 recombinant proteins were incubated with glutathione sepharose (GE Healthcare) in a binding buffer (50 mM Tris, pH 7.4, 120 mM NaCl, 5% glycerol, 0.5% Nonidet P-40, 1 mM PMSF, and 1 mM β-mercaptoethanol), for 2 h at 4°C, then were collected and mixed with supernatant containing His₆-DPA-FLAG and incubated at room temperature for 60 min. After being rinsed five times with washing buffer (50 mM Tris, pH 7.4, 120 mM NaCl, 5% glycerol, and 0.5% Nonidet P-40), the bound proteins were boiled in SDS sample buffer and subjected to SDS-PAGE and immunoblotting.