Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding
David A. Eberhard1,3,*,
Larry R. Karns2,3,
,
Scott R. VandenBerg3 and
Carl E. Creutz1,¶
1 Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
2 Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
3 Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
* Present address: Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
Present address: Argonex Inc., 2044 India Rd. Ste. 202, Charlottesville, VA 22901, USA
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Fig. 1. Localization of AnxII and GFP-AnxII fusion proteins in U1242MG cells. Endogenous AnxII was visualized by indirect immunofluorescence and imaged using epifluorescence photomicrography (AnxII IF). Stably transfected cells expressing EGFP, EGFP-AnxII, EGFP-AnxII(1-22), EGFP-AnxII(23-338) or EGFP-AnxII(L10A/L12A) were fixed and imaged using LSCM. The association of EGFP-AnxII(23-338) with nucleoli is an artifact of fixation, apparently due to residual Ca2+ from the wash buffer. The construct is excluded from nucleoli in living cells (not shown). Bar, 20 µm.
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Fig. 2. Immunoprecipitation of GFP fusion protein complexes. Anti-GFP immunoprecipitation was performed in lysates of stably transfected U1242MG cells expressing EGFP-p11, EGFP-AnxII or EGFP. (Top-left) Western blot for p11; 10% gel transferred to nitrocellulose. The p11 mAb recognized both endogenous p11 and EGFP-p11 in the lysates. Co-immunoprecipitation of p11 was seen only with GFP-p11. (Bottom-left) The same experiment as above; 15% gel transferred to PVDF in buffer containing 30% methanol. This procedure allows separation of p11 from the dye front and maximizes transfer of p11 to the membrane. (Top-right) Western blot for AnxII; 10% gel transferred to nitrocellulose. The AnxII mAb detects EGFP-AnxII only in the immunoprecipitates, since its apparent level in the lysates is much lower than endogenous AnxII. Co-immunoprecipitation of AnxII was seen only with EGFP-p11. (Bottom-right) Coomassie Blue stained gel shows distinct bands corresponding to EGFP and EGFP-AnxII in the immunprecipitates, whereas the much fainter EGFP-p11 reproduced poorly.
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Fig. 3. Ca2+-dependency of GFP-AnxII and GFP-p11 binding to Triton X-100-insoluble cell components. U1242 cells expressing GFP-AnxII or GFP-p11 were extracted with Triton X-100 in the presence of various [Ca2+] as described in Materials and Methods. The soluble and insoluble fractions were subjected to western blot analysis using antibodies against GFP or AnxII. The Ca2+-dependency of endogenous AnxII binding to the insoluble fraction was the same in both cell populations.
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Fig. 4. Temperature dependency of GFP-AnxII(1-22) localization. U1242MG cells expressing GFP-AnxII(1-22) were cooled at 4°C for 30 minutes, then photographed immediately after placement on the microscope stage at RT (left) and again after 10 minutes at RT (right). Warming caused some cells to round up (bottom-left of panels). Bar, 20 µm.
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Fig. 5. GFP-AnxII localization over time after LmB treatment. U1242MG cells expressing GFP-AnxII were treated with LmB for various periods of time before fixation and imaging by LSCM. Nucleic acids were stained with propidium iodide to confirm nuclear localization (not shown). Bar, 20 µm.
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Fig. 6. Effect of LmB on localizations of GFP-AnxII, GFP-p11, AnxII and p11. Stably transfected U1242MG expressing GFP-AnxII (A) or GFP-p11 (B) were treated with LmB for 90 minutes, fixed, processed for indirect immunofluorescence using mAbs against p11 (A) or AnxII (B), and imaged by LSCM. Bars, 20 µm.
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Fig. 7. Effect of LmB on the localization of transiently overexpressed AnxII. U1242MG were transiently cotransfected with pCI-neo-AnxII and pEGFP. Two days after transfection, the cells were incubated with 100 nM LmB or vehicle for 90 minutes, then fixed and processed for indirect immunofluorescence using mAb against AnxII. Bar, 20 µm.
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Fig. 8. Effect of LmB, genistein and pervanadate on AnxII localization in RAT-1(v-src) cells. In (A), cells were incubated for 6 hours in the absence or presence of 100 nM LmB, 100 µM genistein, or vehicle. In (B), cells were incubated for 1 hour in the absence or presence of 100 nM LmB, 3 µM Na pervanadate, or vehicle. Cells were then fixed, processed for indirect immunofluorescence using mAb against AnxII, and imaged by LSCM. Bar, 20 µm.
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Fig. 9. Effect of LmB on co-localization of AnxII and GFP-p11 in RAT-1(v-src) cells. Stably transfected cells expressing GFP-p11 were treated for 5 hours with 100 nM LmB or vehicle, then fixed, processed for indirect immunofluorescence using mAb against AnxII, and imaged by LSCM. Bar, 20 µm.
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