Ub-EEA1 expression induced vacuole-like endocytic structures. A, COS7 cells transfected with the indicated plasmids were immunostained with anti-EEA1 antibodies in red. Cells were counter-stained with DAPI to reveal the nuclei. B, Quantification of Ub-EEA1-induced giant vacuole phenotype. The percentage of cells with enlarged vacuole-like endosomes in either EEA1 or Ub-EEA1 expressing cells is indicated. Error bars, SD (n = 3). C, EEA1- or Ub-EEA1-expressing cells were fixed and analyzed by transmission electron microscopy. The arrow indicates a representative vacuole-like structure in Ub-EEA1-expressing cells. D, Extracts prepared from cells transfected with the indicated plasmids were analyzed by immunoblotting to determine the expression levels of the indicated proteins.