Fig. 3. Mutagenesis of the caspase cleavage site prevents cleavage of the occludin cytoplasmic domain. Ltk^- cells were transiently transfected with either full-length occludin-myc₆ or with occludin (D³²⁰A)-myc₆ with the mutated caspase cleavage site. Apoptosis was induced with 1 µM staurosporine for 3 hours. Western blots of cell lysates (25 µg) were analyzed with monoclonal anti-occludin (clone 19) antibody. Lanes 1-4 control cells, lanes 5-7, cells 3 hours after induction of apoptosis. Lane 1, untransfected Ltk^- cells; lanes 2 and 5, Ltk^- cells transfected with empty vector; lanes 3 and 6, Ltk^- cells transfected with wild-type occludin; lane 4 and 7, Ltk^- cells transfected with mutant occludin (D³²⁰A). Analysis with an anti-myc antibody gives comparable results (not shown). This western blot is a representative of at least five independent experiments.