Fig. 10. (A) Claudin-14 but not the 4S mutant is palmitoylated in rat-1 cells. Tet-off rat-1 cells were stably transfected with VSV-G-tagged claudin-14 and 4S mutant, and expression of the transgene was induced by removal of doxycycline. Cells were labeled with 3H-palmitic acid; immunoblot analysis (top) confirmed expression of both wild-type and mutant claudin-14, and the replicate fluorograph (bottom) demonstrated palmitoylation of wild-type but not mutant claudin-14. (B) Immunofluorescent analysis of rat-1 fibroblasts induced to express wild-type claudin-14 (left) and the 4S mutant (right). Both wild-type and 4S mutant could concentrate at sites of cell-cell contact. Scale bar, 10 µm. (C) Freeze-fracture analysis revealed that both wild-type and mutant claudin-14 form similar patches of tight-junction strands. Claudin-14 and 4S mutant were cultured on glass coverslips, induced for 4 days, fixed with glutaraldehyde and prepared for freeze fracture by a conventional protocol. Electron microscopic freeze-fracture analysis of wild-type (left) and the mutant claudin (right) revealed similar appearing tight-junction strands.