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Fig. 5. Centrosomal targeting of Nlp and ninein depends on dynein-mediated transport. (A) Tet-On U2OS cell lines carrying Myc-tagged Nlp or ninein were treated with nocodazole (left panels) or DMSO (middle panels) for 1 hour before Nlp and ninein expression was induced for 5 hours by addition of tetracycline. Alternatively, Nlp and ninein expression was induced for 4 hours by addition of tetracycline, followed by a 4 hours treatment with nocodazole (right panels). Cells were fixed and then stained with antibodies to the Myc-epitope to detect Nlp and ninein (green), and 
-tubulin (red). (B) U2OS cells treated with nocodazole or DMSO for 4 hours prior to fixation were stained with antibodies to Nlp and -tubulin. (C) Tet-On U2OS stable cell lines carrying Myc-tagged Nlp or ninein were transfected with DsRed-p150 CC1. Addition of tetracycline to the culture medium 12 hours after transfection was used to induce expression of Nlp and ninein. Cells were then grown for 12 hours in the presence of tetracycline, fixed and analysed by immunofluorescence microscopy. Cells transfected with p150 CC1 were detected by DsRed fluorescence (asterisks). Cells were counterstained with anti-Myc antibodies and DNA was stained with DAPI (blue). Bars, 10 µm. A schematic representation of the experimental procedures is shown below each set of images.
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