Screening of RING-type E3 ligases having polyubiquitination activity. A, Polyubiquitination reaction of crude recombinant E3 proteins was carried out with mixture of FLAG-tagged and biotinylated ubiquitins, and investigated by luminescent analysis with anti-FLAG antibody. B, Polyubiquitination activity of At1g55530 was confirmed by immunoblot analysis. The assay was carried out using purified recombinant AtUBC8 and At1g55530, and mobility shift of FLAG-tagged At1g55530 and polymer of His-ubiquitin were detected by immunoblot analysis using anti-FLAG and anti-His antibodies, respectively. Error bars represent standard deviations from three independent experiments.