FIG. 4.

The NTD of SUN1 is responsible for anchoring to the nuclear lamina. (A) Schematic representation of the myc-tagged SUN1 constructs. Labeling is as in Fig. 2A, except that hatched boxes indicate locations of myc tags. (B) NIH 3T3 cells were transiently transfected with myc-SUN1 constructs as indicated and fixed in methanol 24 or 72 h (c, f, h, j, and l) posttransfection. Cells were then costained with anti-myc 9E10 (left side) and 3262 anti-lamin A/C (right side) antibodies. In parts k and l, apparent colocalization of myc-SUN1(450-913) with lamin A/C at the NE is indicated by yellow areas in the merged color images [red, myc-SUN1(450-913); green, lamin A/C]. (C) Transfected NIH 3T3 cells were subjected to pre-extraction with 0.5% Triton X-100 in PBS for 5 min on ice, immediately fixed in methanol, and then subjected to immunofluorescence staining as for panel B. Scale bars, 10 μm. (D) NIH 3T3 cells were transfected with myc-tagged SUN1 constructs encoding the full-length (FL) protein or the NTD (amino acids 1 to 355). Nuclei (Nuc) were isolated and incubated in extraction buffer containing 7 M urea, separated into soluble (Sol) and insoluble (Ins) fractions, and immunoblotted with anti-myc 9E10 antibodies.