EEA1 undergoes mono-ubiquitination in cells. A, Cell extracts prepared from HEK293 cells transfected with HA-tagged ubiquitin- (HA-Ub) and EEA1-FLAG-expressing plasmids (+) or a control empty vector (−) were subject to immunoprecipitation with FLAG antibodies. The precipitated materials were analyzed by immunoblotting (IB) with the indicated antibodies. The bracket indicates ubiquitinated EEA1 species. B, As in A, except that cells expressing HA-Ub were used and that immunoprecipitation was performed with anti-EEA1 antibodies. C, As in B, except that cells expressing the indicated Ub variants were used.