Fig. 2. Ezrin localization at the apical membrane immediately precedes maturation in a population of highly differentiated epithelial cells. (A) Primary culture MTE wild-type cells differentiated for 7 days on semipermeable, supported membranes under ALI conditions were immunostained for ezrin (red) and ß-tubulin-IV (green) expressed in cilia then imaged by immunofluorescent microscopy and merged. Nuclei were detected in the same field by DAPI staining. Bar, 10 µm. (B) Primary culture MTE cells grown and immunostained as in A were harvested on the indicated days (d) and imaged by confocal microscopy at the level of the apical membrane (x,y). Images were reconstructed to generate z-axis images. Bar, 10 µm. (C) MTE cells cultured as in A were partitioned into detergent-soluble and -insoluble fractions as described (Algrain et al., 1993) at indicated days and 10 µg of protein was subjected to immunoblot analysis for detection of ezrin (clone 3C12), ß-catenin (ß-cat) and actin. Representative data from four independent preparations are shown.