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Fig. 2. Nvj1p localizes to the outer leaflet of the nuclear membrane at NV junctions. Immunogold localization of GFP-Nvj1p (A) and Nvj1p-GFP (B) by electron microscopy. Both GFP-tagged Nvj1p reporters were induced in wild-type cells for 1 hour with CuSO4 before cryofixation and immuno-EM analysis. (A) GFP-Nvj1p, a nonfunctional reporter, is mislocalized from NV junctions. Colloidal-gold-conjugated antibodies directed against GFP-Nvj1p localize to the surface of cytoplasmic vacuoles away from the nuclear membrane. Nucleus denoted as N, vacuoles labeled as V. (B) Nvj1p-GFP, a functional reporter, localizes specifically to NV junctions. Colloidal-gold-conjugated antibodies directed against Nvj1p-GFP label the surface of nuclei at NV junctions (i). Higher magnification reveals that most gold particles are preferentially concentrated across from the inner nuclear membrane (ii, inner nuclear membrane outlined in white). Nucleus is denoted as N, vacuoles as V. Scale bars, 0.3 µm.





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