Fig. 3. Rab22a overexpression makes EEA1 resistant to Wortmannin treatment. Wild-type myc-Rab22a was expressed in BHK-21 cells for 14 hours using a recombinant SFV, the cells were then incubated for 30 minutes in the absence (-Wortmannin) or the presence (+Wortmannin) of 100 nM Wortmannin and prepared for double immunofluorescence microscopy with anti-myc and human anti-EEA1 antibodies. In untransfected cells (arrows), EEA1 showed in the absence of Wortmannin (A-B) a characteristic staining of small early endosomal structures and localized in transfected cells on the large vacuole-like Rab22a-positive endosomes. Upon Wortmannin treatment (C,D), a majority of EEA1 was lost from the endosomes of untransfected cells (arrowheads), but intense EEA1 staining remained detectable on the large Rab22a-positive endosomes of the tranfected cells. Bar=10 µm.