Fig. 4. The entire N-terminal domain of MAN1 is necessary for efficient inner nuclear membrane targeting and retention. (A) Proteins expressed by the plasmid constructs MAN8-199-CHL, MAN85-336-CHL and MAN351-CHL, which were used to transfect COS-7 cells. Domains of the proteins are represented by: yellow, FLAG-tag; white, nucleoplasmic domain of MAN1; red, LEM domain; pink, transmembrane segment of CHL; light blue, luminal domain of CHL. Thin lines indicate portions of MAN1 deleted from each protein. The N-terminus of each protein is on the left.

(B) Immunofluorescence images from laser scanning confocal immunofluorescence microscopy showing localization of MAN1-CHL chimeric proteins from which amino acids 8-199 (MAN8-199-CHL), 85-336 (MAN85-336-CHL) and 351-476 (MAN351-CHL) of MAN1 were deleted. 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 to the right show an overlay of the FITC and rhodamine channels, which areas of overlap appearing yellow. Bar, 10 µm.