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Fig. 4. In situ localization of proteasome-dependent protein degradation. Panels (A-C) and (F,G) show confocal fluorescence micrographs of HEp-2 cells with the corresponding differential interference contrast (DIC) image on the left. (A) HEp-2 cells were microinjected with 0.5 mg/ml fluorogenic substrate protein DQ-OVA into the cytoplasm and cell nucleus (cell a) or into the cell nucleus (cell b) or into the cytoplasm (cell c). Microinjected DQ-OVA shows a granular cytoplasmic localization. In the cell nucleus DQ-OVA localizes to distinct nucleoplasmic foci. Unlabelled areas correspond to nucleoli. Note, DQ-OVA is excluded from the nucleus in cells that were exclusively microinjected into the cytoplasm. (B) A representative HEp-2 cell that was microinjected with DQ-OVA and 10 µM proteasome inhibitor lactacystin shows a diffuse localization of DQ-OVA in the nucleus without formation of nucleoplasmic foci. (C) Equal numbers of HEp-2 cells in highlighted areas were microinjected with DQ-OVA (–LC) or DQ-OVA and 10 µM lactacystin (+LC). Corresponding fluorescence micrographs show decreased DQ-OVA fluorescence in proteasome inhibitor-treated (+LC) versus untreated (–LC) cells. (D) Fluorescence-quantification of (C). (E) HEp-2 cells were treated as in (C), and fluorescence of DQ-OVA quantified after 5, 10 and 15 minutes (min). (F) HEp-2 cells were co-incubated for 30 minutes with 200 µg/ml DQ-OVA, which localizes to endosomal vesicles in the cytoplasm, whereas the nucleus is unlabelled. (G) Confocal immunofluorescence of a representative HEp-2 cell that was microinjected with 200 µg/ml plain ovalbumin, fixed and immunolabelled with anti-ovalbumin antibodies. The ectopic protein ovalbumin distributes diffusely throughout the cytoplasm, and cell nucleus. Scale bars, 10 µm (A,B,F,G); 150 µm (C).
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