Detection of polyubiquitination and self-ubiquitination of CIP8. A to D, The polyubiquitination assay was carried out with purified (A and B) or crude recombinant CIP8 (C and D) and detected by luminescent analysis with anti-FLAG antibody (A and C) and immunoblot analysis (B and D). His-Ub or Mix-Ub indicate His-tagged ubiquitin or the mixture of FLAG-tagged and biotinylated ubiquitin, respectively. The polyubiquitination assay using luminescent analysis was carried out with recombinant CIP8 without tag in the presence or absence of ubiquitin related components indicated below each graph. E, Ubiquitination of crude recombinant CIP8 was observed by luminescent analysis with anti-FLAG antibody. The assay was carried out with or without biotinylated ubiquitin and crude AtUBC8 recombinant protein. Bio-Ub means biotinylated ubiquitin. Error bars represent standard deviations from three independent experiments.