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Fig. 2. Giantin and syntaxin 5 are rapidly cleaved during apoptosis in vivo. (A) HeLa cells were treated with 1 µM staurosporine in the absence or presence of 50 µM zVAD.fmk (Z) for the times indicated and analysed by immunoblotting with antibodies to PARP, giantin, GM130, syntaxin 5 and GS28. (B) HeLa cells were treated with anisomycin for 12 hours. Apoptotic cells became detached from the culture dish and these floating cells (F) were harvested by centrifugation. Remaining adherent cells (A) were harvested in parallel, and samples from each population were analysed by immunoblotting with antibodies to PARP, giantin, GM130, syntaxin 5 and GS28. Full-length (FL) and caspase cleavage products (*) are marked. The major giantin cleavage products P1-P3 are shown. Minor cleavage products are indicated with filled circles. (C) Confocal images of HeLa cells treated with UV radiation to induce apoptosis. Cells were incubated for 4 hours post-irradiation and stained with DAPI (blue), antibodies to GM130 (green) and antibodies raised against either the N terminus of giantin (NT) or fragments encompassing the entire protein (mix; red). Regions of overlap between GM130 and giantin are in yellow. Apoptotic cells are marked with arrowheads. Scale bar, 10 µm.
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