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Fig. 2. Effect of long-term TNF
treatment on MLC phosphorylation state in TtT/GF cells. TtT/GF cells were incubated with medium alone or with medium containing 20 ng/ml TNF for 96 hours. Following the treatments, cells were scraped off and total-cell lysate (T), non-cytoskeleton (N) and cytoskeleton (C) fractions were prepared and subjected to electrophoresis and western blotting with different antibodies. (A) Specific antibody against MLC phosphorylated at Ser19 [labelled P-(Ser19)-MLC] detected phosphorylated MLC in the cytoskeleton-enriched (C) fraction of control and treated cells. TNF increased MLC-Ser19-P levels. The increase was blocked by the caspase inhibitor Z-VADfmk. The same membrane was stripped and reprobed for MLC. Most MLC (arrow) was recovered in the cytoskeleton fraction. Cytoskeleton-associated MLC was slightly increased by TNF, whereas non-cytoskeleton-associated MLC decreased in TNF-treated cells. The decrease was partly abolished by the caspase inhibitor Z-VAD-fmk. The actin immunoreactive bands correspond to the loading control. (B) Incubation of TtT/GF cells with TNF for 96 hours induced ROCK1 cleavage and translocation to the cytoskeleton fraction of the full-length enzyme. Cleaved, active ROCK1 was recovered in the cytoskeleton-enriched fraction (arrowhead). The caspase inhibitor Z-VAD-fmk blocked TNF-induced ROCK1 cleavage. The actin immunoreactive bands correspond to the loading controls. (C) Representative phase-contrast micrographs of TtT/GF cells incubated with culture medium alone or with medium containing TNF both in the absence (control) or presence of the ROCK inhibitor Y-27632 (5 µM). ROCK inhibition in control cells caused cell-body retraction and increased lamellipodia and cellular processes. TNF-induced cell shrinkage and detachment were abolished by Y-27632. Scale bar: 100 µm.
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