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Fig. 1. Occludin is cleaved during apoptosis of epithelial cells. (A) H184A1 cell lysates (35 µg) were analyzed by western blotting with the monoclonal anti-occludin (clone 19) antibody at different times after induction of apoptosis with staurosporine. Two specific occludin fragments (fragment 1 and 2) were generated with time. The middle panel shows a shorter exposure of the above western blot to show the decrease in full-length occludin more clearly. The lower panel shows the degradation of PARP in the same cell lysates that were used to analyze occludin cleavage. (B) The generation of fragment 1 and decrease of full-length occludin were quantified by chemiluminescence imaging on a FujiFilm LAS-1000 system. The signal for occludin at 0 hours and the strongest signal for fragment 1 at 12 hours were set to 100%. Data represent mean values of five independent experiments with A as a representative blot. (C) Apoptosis was induced by addition of TNF-{alpha} for 24 hours. Cell lysates (15 µg) of control (lane 1), adherent (lane 2) and floating apoptotic MDCK cells (lane 3) were analyzed using western blots with the monoclonal anti-occludin (clone 19) antibody. (D) After 6 hours pretreatment of cells with IFN-{gamma}, apoptosis was induced by addition of anti-CD95/Fas (clone CH11) antibody for 24 hours and western blot analysis of lysates (20 µg) of control (lane 1), adherent (lane 2) and floating H184A1 cells (lane 3) was performed with monoclonal anti-occludin (clone 19) antibody. Western blots in C and D are representatives of at least four independent experiments. (E) Measurement of transepithelial resistance Rt in HT-29/B6 cells seeded on Millicell filters and incubated with 104 U/ml TNF-{alpha} alone and TNF-{alpha} in the presence of 50 µM Z-VAD-FMK. Rt was monitored over a period of 12 hours and specified as a percentage of initial Rt values. The results are representative of at least three independent experiments (Student's t-test was used to compare results, **P<0.01).





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