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Figure 3


Fig. 3. Nesprin-3{alpha} is dependent on Sun1 and Sun2 for its localization at the NE. (A) PA-JEB cells stably expressing GFP-nesprin-3{alpha} were mock transfected or transfected with siRNA directed against lamin A/C (LmnA), Sun1, Sun2 or a combination of Sun1 and Sun2. 72 hours after transfection, cells were lysed and WCLs were analyzed for expression of the indicated proteins. (B) PA-JEB cells stably expressing GFP-nesprin-3{alpha} were mock transfected or transfected with siRNAs directed against LmnA, Sun1, Sun2 or a combination of Sun1 and Sun2. 72 hours after transfection, cells were fixed in paraformaldehyde, stained for the indicated proteins and analyzed by confocal microscopy. Bar, 10 µm. (C) Quantitative analysis of the effect shown in (B). The gray values of GFP-nesprin-3{alpha} at the nucleus and the complete cell were determined for individual siRNA- and mock-transfected cells. Results are shown as the mean ratio of the gray value in the nucleus to the gray value in the total cell±s.e.m. (n=50). *, P<0.001. (D) PA-JEB cells stably expressing GFP-nesprin-3{alpha} were transiently transfected with a Myc-tagged Sun2 lumenal domain construct carrying an ER-retention signal (Sun2L-ER). After 72 hours, cells were fixed in paraformaldehyde and stained for Myc. Representative confocal images are shown. Arrows indicate a displacement of GFP-nesprin-3{alpha} over the ER. Bar, 10 µm.





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