Fig. 3. The nucleoplasmic, N-terminal domain of MAN1 plus its first transmembrane domain is localized to the inner nuclear membrane using digitonin-permeabilized cells. (A) Protein expressed by the plasmid construct MAN538-GFP, which was used to transfect COS-7 cells. Domains of the protein are represented by: yellow, FLAG-tag; white, nucleoplasmic, luminal and C-terminal domains of MAN1; red, LEM domain; orange, the first transmembrane segment of MAN1; green, GFP. The N-terminus of the protein is on the left. (B) Fluorescence microscopic analysis of COS-7 cells that were transfected with plasmid expressing the FLAG-tagged N-terminal domain and first transmembrane segment of MAN1 fused to GFP. The cells were permeabilized with Triton X-100 (upper panels, labeled Triton X-100) or ice-cold digitonin (lower panels, labeled Digitonin) and were then labeled with anti-FLAG monoclonal antibody. The left panels (GFP) show fluorescence from GFP (green), the middle panels (FLAG) show signal from anti-FLAG monoclonal antibodies recognized by rhodamine conjugated secondary antibodies (red) and the right panels (Overlay) show an overlay of the GFP and FLAG panels, with areas of overlap appearing yellow. Bar, 10 µm.

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