Fig. 5. Size of the nucleocytoplasmic domain of MAN1 affects its inner nuclear membrane targeting. (A) Protein expressed by the plasmid CMPK-MAN538, which was used to transfect COS-7 cells. Domains of the protein are represented by: yellow, FLAG-tag; dark blue, truncated CMPK; white, nucleoplasmic domain of MAN1; red, LEM domain; orange, the first hydrophobic transmembrane segment of MAN1. The N-terminus of the protein is on the left. (B) Immunofluorescence images from laser scanning confocal immunofluorescence microscopy showing localization of truncated CMPK fused to the N-terminal domain and first transmembrane segment of MAN1. Antibodies were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (green, upper and lower left panels labeled FLAG), polyclonal antibodies against lamin B1 (labeled Lamin B1) or against SSR-alpha (labeled SSR), which is a marker for ER protein, recognized by rhodamine-conjugated secondary antibodies (red, middle panels). The right panels show an overlay of the FITC and rhodamine channels, with areas of overlap appearing yellow. Bar, 10 µm.

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