EEA1 can interact with E2 to undergo E3-independent mono-ubiquitination. A, In vitro ubiquitination was performed with a post-nuclear extract isolated from cells expressing EEA1-FLAG in the presence of HA-tagged ubiquitin and ATP as indicated. The reactions were analyzed by dual color immunoblotting with the indicated antibodies. B, A Coomassie-blue stained gel showing the purified EEA1 (indicated by the arrow). The bracket indicates 3XFLAG peptide. C, Purified EEA1 was incubated with the indicated E2 ubiquitin conjugating enzymes together with E1, HA-Ub, and ATP. Where indicated, EEA1-FLAG was omitted as negative controls. The reactions were analyzed by immunoblotting with the HA (top panel) and EEA1 antibodies (bottom panel). D, As in A, except that the indicated ubiquitin variants were used. E, F, Interaction of EEA1 with Ube2A. E, Purified Ube2A was injected into a CM5 chip immobilized with EEA1-FLAG at the indicated concentrations. The interaction of Ube2A with EEA1 was monitored by Surface Plasmon Resonance. F, The interaction of EEA1-FLAG with Ube2A was plotted against the E2 concentrations.