Ae016

MBP-BES1 was used in our previous study (Zhang et al., 2014) and purified using amylose resin (NEB, Ipswich, MA, USA). UBP12 and UBP13 were cloned into the pET-28a (+) vector (His tag) and purified using NiNTA agarose (Invitrogen, Carlsbad, California, USA). Purified MBP, MBP-BES1 were incubated with equal amounts of His-UBP12 or His-UBP13 beads in His pull-down binding buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% Triton) at 4°C for 2 h. After washing 7 times with His pull-down washing buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton), the beads were collected, boiled in 50 μL 2 × SDS loading buffer for 8 min at 100°C, and the sample examined by immunoblotting using anti-His (1:5,000 dilution, ABclonal, China, AE003) and anti-MBP (1:5,000 dilution, ABclonal, AE016) antibodies. The antibodies source details are listed in Supplemental Table S2. The primers used for plasmid construction are listed in Supplemental Table S1.

In vitro pull-down assays were performed as described previously (Zhang et al. 2021) . Full-length HLS1 and SIZ1 were cloned into pMAL-c2x (MBP tag) and pET-28a (+) vector (His tag), respectively (Zhang et al. 2021) . MBP-HLS1 was purified using amylose resin (NEB), and His-SIZ1 was purified using NiNTA agarose (Invitrogen). Purified MBP or MBP-HLS1 beads were incubated with equal amounts of His-SIZ1 in MBP pulldown binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA) at 4 °C for 2 h. After washing 7 times with MBP pull-down washing buffer [200 mM NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet P-40, 20 mM Tris-HCl, pH 8.0], the beads were collected, boiled in 50 μL 2× SDS loading buffer for 8 min at 100 °C, and the sample examined by immunoblotting using anti-His (1:5,000 dilution, ABclonal, China, AE003) and anti-MBP (1:5,000 dilution, ABclonal, China, AE016) antibodies. The primers used for plasmid construction are listed in Supplemental Data Set 1.