Fig. 2. The N-terminal domain of MAN1 contains a nuclear envelope targeting signal. (A) Proteins expressed by the plasmid constructs MAN538, MAN476-CHL and CT, which were used to transfect COS-7 cells. Domains of proteins 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; dark green, the second transmembrane segment of MAN1; pink, transmembrane segment of CHL; light blue, luminal domain of CHL. The N-terminus of each protein is on the left. (B) Cellular localizations of the N-terminal domain with the first transmembrane segment of MAN1 (MAN538), the N-terminal domain of MAN1 (amino acids 1-476) fused to truncated CHL (MAN476-CHL) and the two transmembrane segments and nucleoplasmic, C-terminal domain of MAN1 (CT) as determined by laser scanning confocal immunofluorescence microscopy. Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (green, left panels labeled FLAG) and polyclonal antibodies against lamin B1 recognized by rhodamine-conjugated secondary antibodies (red, middle panels labeled Lamin B1). The panels on the right show an overlay of the FITC and rhodamine channels, with areas of overlap appearing yellow. Bar, 10 µm.

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