Analysis of recombinant Arabidopsis HECT-type E3 ligases (UPL7 and UPL5). A, Production of FLAG-tagged recombinant UPL proteins was detected by immunoblot analysis. For analysis, 5 μl of crude recombinant UPL proteins were loaded, and detected by immunoblot analysis using anti-FLAG antibody. B, Schematic diagram of detection of ubiquitin-conjugation of UPLs by luminescent analysis. Protein A-conjugated acceptor beads and streptavidin-coated donor beads were bound to anti-FLAG antibody bound to FLAG-tagged recombinant UPLs and biotinylated ubiquitin, respectively, and detected by same principle and procedure described in Figure 1B. C, The ubiquitination of crude recombinant UPL7 and UPL5 was detected by luminescent analysis described in B. Bio-Ub means biotinylated ubiquitin. D, polyubiquitination of crude recombinant UPL7 and UPL5 was detected by luminescent analysis with anti-His antibody. Mix-Ub indicated the mixture of His-tagged and biotinylated ubiquitin. E and F, Mobility shift of UPLs (E) and formation of polyubiquitin chains (F) were detected by immunoblot using anti-FLAG antibody and Alexa488-conjugated streptavidin, respectively. The polyubiquitination reaction was done with FLAG-tagged recombinant UPLs in presence or absence of crude AtUBC8, and then recombinant UPLs were purified by anti-FLAG antibody-conjugated agarose. Error bars represent standard deviations from three independent experiments.