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Fig. 2. Fragment 1 and 2 are generated by caspase and metalloproteinase cleavage, respectively. Western blots in A, B and C are representative of at least four independent experiments. (A) MDCK cell lysates (10 µg) were analyzed 6 hours after induction of apoptosis by staurosporine in the presence or absence of Z-DEVD-FMK with the monoclonal anti-occludin (clone 19) antibody. (B) Apoptosis was induced by 24 hours TNF-
treatment and equal amounts of protein from lysates (15 µg) of MDCK cells were analyzed in the presence of caspase and/or metalloproteinases inhibitors (TAPI-2, 50 µM; MMPI-1, 100 µM) using the monoclonal anti-occludin (clone 19) antibody. (C) In vitro cleavage of GST-occludin264-522 by recombinant caspase-3 generates a cleavage fragment co-migrating with fragment 1 from H184A1 cell lysates. In vivo cleavage of occludin in H184A1 cells at 0 hours (lane 1) and 12 hours (lane 2) after induction of apoptosis. In vitro cleaved GST-occludin264-522 (lane 3). Western blots were analyzed with the polyclonal anti-occludin antibody. (D) Schematic model of occludin and the apoptotic cleavage sites.
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