The pre-treated OsPUB15 possesses E3 ligase activity. (A) E3 ligase activity analysis of pre-incubated His-OsPUB15. The purified His-OsPUB15 was incubated with total protein extracts of rice for one hour, and then was put into self-ubiquitination assays in the presence of wheat E1, human E2 (UBCh5b) and 6xHis tag ubiquitin (His-Ub) or in the absence of E1, E2 or His-OsPUB15 (lanes 5-8). As a control, the un-pre-incubated His-OsPUB15 was included in the assays (lanes 1-4). After the reactions, samples were separated by 12% SDS-PAGE and immunoblotted with anti-Ub (top panel) or anti-OsPUB15 (bottom panel) antibody. (B) E3 ligase activity analysis of PID2K-phosphorylated His-OsPUB15. The untreated His-OsPUB15 (lanes 1-4) and the PID2K-phosphorylated His-OsPUB15 (lanes 5-8) were assayed for ubiquitination activity, respectively. The reaction products were immunoblotted with anti-Ub (top panel) or anti-OsPUB15 (bottom panel) antibody. These experiments were repeated three times with similar results obtained.