Fig. 4. Foxj1 regulates apical localization of ezrin in primary culture airway epithelial cells. (A) Wild-type (+/+) or Foxj1-null (–/–) MTE cells were cultured under ALI conditions and at ALI day 14 were immunostained for expression of ezrin (red) and ß-tubulin-IV (green) as in Fig. 2. Null MTE cells (right) were transfected with an adenovirus vector expressing Foxj1 or control GFP (not shown) at ALI day 0, then harvested at ALI day 14. Identical fields were imaged for each genotype and condition. Bar, 30 µm. (B) Primary culture MTE cells were collected at ALI day 14. Cells analyzed in the upper panel were separated by centrifugation into supernatant (cytosol portion) and pellet (membrane-cytoskeleton portion) as described (Parlato et al., 2000) and 2 µg of protein analyzed per lane. Samples in the lower panel were separated by Triton X-100 detergent solubility into soluble fraction (cytosol portion) and insoluble fractions (membrane-cytoskeleton portion) as described (Algrain et al., 1993). Proteins (4 µg) were subjected to immunoblot analysis for detection of ezrin (clone 3C12) and actin. (C) Relative protein expression ratio of ezrin to actin in the membrane-cytoskeletal portion determined by densitometry from immunoblots as in B. Data represent the mean and standard deviation of expression from three independent preparations. The asterisk indicates a difference in means (P<0.05).