Ni nta agarose beads

The 14-day-old Arabidopsis seedlings were ground in liquid nitrogen and suspended in 1 mL extraction buffer (containing 50 mM PBS pH 7.0, 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, and 1× proteinase inhibitor cocktail). Protein extracts of WT plants were centrifuged at 12,000 g at 4°C for 30 min for later experiments. The expression and purification of the His-TF-AtPRMT5 protein were performed with Ni-NTA agarose beads (Sangon, China) in a 10-mM HEPES buffer using an ultrafiltration device. After ultrafiltration, the His-TF-AtPRMT5 recombinant protein was bound with pre-treated Ni-NTA agarose beads for 1 h. The unbound proteins were removed by washing three times with solution A (20 mM Tris-Cl, pH 7.5), and then, protein extracts of WT were incubated with Ni-NTA agarose beads at 4°C for 20 min. The beads were collected and washed three times with washing buffer (20 mM Tris-Cl, pH 7.0, 0.5 mol/L NaCl) to remove unbound proteins. The resuspended immunoprecipitates were separated using 12% SDS-PAGE and exposed to an anti-AtLCD antibody (2,000×).