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Fig. 6. Cleavage of ZO-1 and ZO-2 in apoptotic H184A1 cells. (A) Apoptosis was induced either by staurosporine (lanes 2 and 3) for 12 hours or anti-CD95/Fas (clone CH11) antibody (lanes 4 and 5) for 24 hours. Lysates (10 µg) of adherent (lanes 2 and 4) and floating (lanes 3 and 5) cells were analyzed by western blotting with a polyclonal anti-ZO-1 antibody. Lane 1, control at 0 hours. (B) Western blot analysis of ZO-2 24 hours after induction of apoptosis with the anti-CD95/Fas (clone CH11) antibody. Lane 1, control at 0 hours; lane 2, adherent cells; lane 3, floating cells; lane 4, lysate of pooled adherent cells and remaining floating cells in the presence of inhibiting anti-CD95/Fas (clone ZB4) antibody. Both western blots are representative of at least four independent experiments. (C,D) Apoptosis was induced by staurosporine and 10 µg of lysates of adherent (lane 2) and floating (lane 3) cells were analyzed by western blotting with anti-ZO-1 (C) or anti-ZO-2 (D) antibody. In the presence of Z-DEVD-FMK cells remain attached and fragmentation of ZO-1 and ZO-2 is inhibited (lane 4). Lane 1, control at 0 hours. Arrows in A-D indicate ZO-1 and ZO-2 cleavage products.
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