Ubch5c

GST-ZmRFWD3, S-tag-O2, and mutations of O2 at Lys residues were overexpressed and purified from E. coli (DE3) Rosetta cultures using standard protocols. For in vitro ubiquitination assays, reaction mixtures (20 mL) contained 0.25 mg of human E1 enzyme (cat. no. UB101; Life-Sensors), 0.2 mg of purified human E2 enzyme UBCH5C (cat. no. UB201; LifeSensors), 0.5 mg of purified GST-tagged ZmRFWD3 protein as the E3 ubiquitin ligase, 0.2 mg of purified S-tag-O2 as substrate, and 1 mg of ubiquitin (Sigma-Aldrich) in 25 mM of Tris-HCl at pH 7.5, 1 mM of MgCl 2 , 1 mM of ATP, and 0.5 mM of DTT. Reactions were incubated at 30°C for 3 h and stopped by adding Laemmli buffer and boiling for 10 min. ubiquitination was detected by anti-S-tag antibody at a dilution of 1:5,000 (cat. no. ab183674; Abcam). A self-ubiquitination assay was performed using the same protocol without S-tag-O2, and GST-ZmRFWD39s self-ubiquitination was detected with anti-GST antibody at a dilution of 1:5,000 (cat. no. ab19256; Abcam). The anti-S-tag and anti-GST tag antibodies were detected using goat anti-rabbit IgG conjugated to horseradish peroxidase at a dilution of 1:5,000 (cat. no. 12-348; Sigma-Aldrich).