In vivo Ubiquitination Assay for BES1

In vivo ubiquitination assays were performed using 7-day-old WT (Col-0), BES1^OE, BES1^OE/UBP12^OE, and BES1^OE/ubp12-2w/13-3 plants. Seedlings were treated with 1 μM eBL for 6 h. Proteins were extracted with 2× protein extraction buffer as described by Yang et al. (2017) (link) (100 mM Tris–HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA [pH 8.0], 1% Triton X-100, 10% glycerol, 50 μM MG132, and protease inhibitor), and extracts were centrifuged at 12 000 rpm for 10 min at 4°C. After pre-clearing for 1 h, total soluble protein extracts were incubated with TUBE magnetic beads (LifeSensors, cat. no. UM-0402M) at 4°C for 4 h. The magnetic beads were washed 4 times with 1× protein extraction buffer. The pooled protein mixtures were separated by SDS–PAGE and analyzed by immunoblotting with anti-myc antibody (Proteintech, cat. no. 16286-1-AP) and anti-Ubi antibody (Santa Cruz Biotechnology, P4D1, cat. no. sc-8017). All assays were repeated in three independent experiments.