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Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits
Mol. Biol. Cell Werner et al. 10.1091/mbc.E03-09-0687.

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Figure 11 Dependence of keratin IF from MT. Double fluorescence analyses of wt and mut MCF-7 cells with an anti-tubulin antibody after the depolymerization of MT by the application of 40 然 nocodazole for 5 h (A, B), or after subsequent regeneration of the cytoskeleton in nocodazole-free culture medium for 1 h (C-F). A, E: EYFP-K14 fluorescence; C-D: indirect immunofluorescence of tubulin; the arrows mark MT growing from centrosomes. B, F: merged images of indirect immunofluorescence of MT (red), EYFP-K14R₁₂₅C (green), and DNA stained by DAPI (blue) in MCF-7 cells; the arrow marks the complete dissociation of a metaphase spindle; Scale bar 10 痠. G: Total protein extracts (T), insoluble cytoskeletal (C), and soluble fractions (S) were prepared from parental MCF-7 (PAR), EYFP-K14- (WT), and EYFP-K14R₁₂₅C (MUT) cells after incubation with 40 然 nocodazole for 3 h, blotted onto membranes, and analyzed with antibodies to GFP and K14.

QuickTime Videos

movie01.mov (9.93 MB) Time lapse recording of EYFP-K14R₁₂₅C cells (30 s recording intervals). To avoid escape of structures from focus at each time point a stack of 6 planes was recorded and projected into a single image.

movie02.mov (1.72 MB) Time lapse recording of the cell edge of an EYFP-K14R₁₂₅C cell (30 s recording intervals). For better visualization an inverse presentation was chosen. The gray line represents the cell edge that was edited from parallelly recorded bright field images.

movie03.mov Time-lapse recordings of EYFP-K14R₁₂₅C cells after treatment with 40 然 nocodazole. The recording (90 s recording intervals) starts 15 minutes after application of the drug.

movie04.mov (3.33 MB) Time-lapse recordings of EYFP-K14R₁₂₅C cells after treatment with 10 然 latrunculin B. The recording starts 13.5 minutes after application of the drug (30 s recording intervals).

movie05.mov (7.92 MB) Recording of EYFP-K14R₁₂₅C cells before and after treatment with 200 然 cytochalasin D (30 s recording intervals).

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