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Fig. 8. Effect of TNF
+CHX on PKC
cleavage. (A) TtT/GF cells were incubated with TNF+ CHX for increasing periods of time. Next, total cell lysates were prepared and subjected to western blotting with antibodies to PKC and active caspase 3. The membrane was striped and incubated with anti-actin as loading control. The representative western blots shows PKC cleavage in cell incubated with TNF+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+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. The figure shows representative western blots. PKC is present in both subcellular fractions in control and treated cells. TNF+CHX treatment induced the translocation of PKC from the non-cytoskeleton (N) to the cytoskeleton (C) fraction. The PKC cleavage product was only found in the cytoskeleton (C)fraction of TNF+CHX-treated cells. The caspase inhibitor Z-VAD-fmk blocked TNF+CHX-induced PKC cleavage but not its translocation. (C) TtT/GF cells, NIH 3T3 fibroblasts, wild-type and MHCIIB–/– embryonic fibroblasts were treated with TNF+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. The representative western blots show the appearance of a 48 kDa immunoreactive band that corresponds to one of the cleavage products of PKC after TNF+CHX treatment in all cell lines.
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