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Nuclear translocation of ferritin in corneal epithelial cells

Cindy X. Cai and Thomas F. Linsenmayer*

Department of Anatomy and Cellular Biology, Tufts University Medical School, Boston, MA 02111



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  		Fig. 1. Micrographs of primary cultures of day-14 corneal epithelial cells. (A) A low power phase-contrast micrograph of a portion of an epithelial sheet. (B) A fluorescence micrograph showing epithelial cells reacted with anti-chicken ferritin antibody 6D11. (C and D) The same cells transfected with a myc-tagged, full-length ferritin construct visualized by (C) Hoffman interference and (D) immunofluorescence with an anti-myc antibody, 9E10. The arrow in (B) points to a nucleolus. The asterisks in (C) and (D) demarcate the same two nuclei. Bar, 25 µm (A); Bar, 10 µm (B-D).

 


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  		Fig. 2. The structural domains of the ferritin-H chain plus the series of deletion constructs of myc-tagged ferritin chain. The top shows a diagram of the structural domains (five {alpha} helices and the loop region) of the wild-type ferritin-H (WT), based on similarities to human ferritin-H (Harrison and Arosio, 1996b). Along the left side are the names of the constructs and their ability to undergo supramolecular assembly (+ or -). Along the right side are their subcellular localizations when transfected into day-14 corneal epithelial cells (CE) and corneal fibroblasts (CF). ‘N’ and ‘C’ indicate their nuclear and cytoplasmic localizations.

 


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  		Fig. 3. Immunofluorescence micrographs of transfected day-14 corneal epithelial cells reacted with anti-myc antibody 9E10. The transfected constructs in the top row (HM, D11 and D13) showed predominantly nuclear immunoreactivity. The constructs in the middle row (D10, DL and DD) showed predominantly cytoplasmic immunoreactivity; however, these constructs were capable of undergoing nuclear translocation if the NLS of SV-40 large T antigen was added to them, yielding the bottom row (D10-NLS, DL-NLS, DD-NLS). Bar, 10 µm.

 


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  		Fig. 4. Immunoblots of non-denaturing gels of lysates from corneal epithelial cells transfected with those constructs that localize to the nucleus (HM, D14, D13 and D11) and to the cytoplasm (D10 as an example), reacted with anti-myc antibody 9E10. Construct HM produced a protein band migrating similarly to the endogenous ferritin at ~260 kDa. The high molecular weight bands were also observed in cells transfected with D14 and D13. Some low molecular weight bands migrating at ~22 kDa were seen in D14- and D13-transfected cells, showing the monomeric myc-tagged ferritin. Only the low molecular weight bands were seen in D11- and D10-transfected cells. The faint high molecular weight band seen in D11 (arrow) was from the nonspecific binding of the secondary antibody (see text).

 


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  		Fig. 5. Immunofluorescence micrographs of transfected corneal fibroblasts reacted with anti-myc antibody 9E10. Fibroblasts transfected with constructs HM and D13 showed predominantly cytoplasmic signals for myc-tagged ferritin, whereas cells transfected with construct D11 showed both nuclear and cytoplasmic signals (top row). When the NLS from the SV-40 large T antigen was attached to these constructs, yielding HM-NLS, D13-NLS and D11-NLS, their products underwent nuclear translocation (bottom row). Bar, 10 µm.

 

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© The Company of Biologists Ltd 2001