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Figure 8


Fig. 8. Effect of TNF{alpha}+CHX on PKC{zeta} cleavage. (A) TtT/GF cells were incubated with TNF{alpha}+ CHX for increasing periods of time. Next, total cell lysates were prepared and subjected to western blotting with antibodies to PKC{zeta} and active caspase 3. The membrane was striped and incubated with anti-actin as loading control. The representative western blots shows PKC{zeta} cleavage in cell incubated with TNF{alpha}+CHX for more than 16 hours. The cleavage was coincident with caspase 3 activation. (B) TtT/GF cells were incubated culture medium alone or containing TNF{alpha}+CHX either in the absence or presence of the caspase inhibitor Z-VAD-fmk for 16 hours. Next, the cells were homogenised and non-cytoskeleton (N)- and cytoskeleton (C)-enriched fractions were prepared. The subcellular fractions were subjected to western blot analyses with anti-PKC{zeta}. The figure shows representative western blots. PKC{zeta} is present in both subcellular fractions in control and treated cells. TNF{alpha}+CHX treatment induced the translocation of PKC{zeta} from the non-cytoskeleton (N) to the cytoskeleton (C) fraction. The PKC{zeta} cleavage product was only found in the cytoskeleton (C)fraction of TNF{alpha}+CHX-treated cells. The caspase inhibitor Z-VAD-fmk blocked TNF{alpha}+CHX-induced PKC{zeta} cleavage but not its translocation. (C) TtT/GF cells, NIH 3T3 fibroblasts, wild-type and MHCIIB–/– embryonic fibroblasts were treated with TNF{alpha}+CHX for increasing periods of time. Next, cell lysates were prepared and 10 µg protein per sample were subjected to electrophoresis and western blotting with anti-PKC{zeta}. The representative western blots show the appearance of a 48 kDa immunoreactive band that corresponds to one of the cleavage products of PKC{zeta} after TNF{alpha}+CHX treatment in all cell lines.