Fig. 8. Overexpression of the C-terminal sequence of JAM retards the restoration of tight junction formation in confluent MDCK II cells after a Ca²⁺ switch. MDCK II cells were transfected with plasmids encoding the JAM mutants as described in the legend to Figure 7. Having reached confluency, cells were subjected to a Ca²⁺ switch. Two or four hours after the return to normal Ca²⁺ concentration, cells were fixed for immunofluorescence analysis. (A) Statistical analysis of cell numbers showing incomplete junctional staining of ASIP. Cells expressing JAM mutants that were identified by anti-Flag immunostaining were categorized into incomplete (black bars) or complete (grey bars) based on their ASIP junctional staining, or aberrant (white bars) based on cell shape. Data represent mean values of three independent experiments with a minimum of 200 cells counted for each sample. (B) Typical immunofluorescent images of cells expressing JAM mutants 2 h after a Ca²⁺ switch. Cells were stained with anti-Flag and anti-ASIP antibodies as indicated. Bar, 15 µm.