Lussi YC, Hügi I, Laurell E, Kutay U, Fahrenkrog B.

	FIGURE 1:. Lamin A is a novel interaction partner of Nup88. Bacterially expressed GST-Nup88 and GST were bound to glutathione sepharose beads and incubated with HeLa S3 nuclear extract. Bound fractions were analyzed by SDS–PAGE, colloidal blue staining, and mass spectroscopy (A) and immunoblotting using anti–lamin A antibodies (B). Lamin A was bound to GST-Nup88, but not to GST. (C) HeLa cell lysates were immunoprecipitated with anti-Nup88 antibodies, and the lysate (L), immune supernatant (S), and immune precipitate (P) were analyzed by SDS–PAGE and immunoblotted with anti-Nup88 and anti–lamin A antibodies. Lamin A coimmunoprecipitated with Nup88.
	FIGURE 2:. The N-terminal domain of Nup88 is interacting with the tail domain of lamin A. (A) Blot-overlay assay employing bacterially expressed and purified vimentin and lamin A and in vitro expressed and 35S-labeled Nup88. Samples of purified vimentin and lamin A were separated by SDS–PAGE, stained with Coomassie blue (left panel), or transferred to a PVDF membrane and overlaid with 35S-labeled Nup88 (right panel). 35S-labeled Nup88 was visualized by autoradiography. (B) Bacterially expressed his-tagged lamin A was bound to nickel beads and incubated with in vitro synthesized 35S-labeled, full-length, N-terminal (residues 1–550) or C-terminal (residues 551–741) Nup88. Unbound and bound fractions were analyzed by SDS–PAGE and autoradiography. 35S-labeled, full-length Nup88 and N-terminal 35S-Nup88 were binding to lamin A-his, whereas C-terminal 35S-Nup88 was not able to bind lamin A-his. (C) Blot-overlay assay using bacterially expressed and purified lamin A and lamin A fragments and in vitro expressed and 35S-labeled Nup88. Samples of purified lamin A were separated by SDS–PAGE, stained with Coomassie blue (left panel), or transferred to a PVDF membrane and overlaid with 35S-labeled Nup88 (right panel). 35S-labeled Nup88 was visualized by autoradiography. (D) Blot-overlay assay using bacterially expressed and purified tail domains of lamin A, lamin B1 and B2, and bacterially expressed and purified GST-Nup88 as well as GST alone. Samples of purified tail domain of lamin A (LA tail), lamin B1 (LB1 tail), and B2 (LB2 tail) were separated by SDS–PAGE, transferred to a PVDF membrane, stained with Ponceau S (left panel), and overlaid with purified GST-Nup88 and GST, respectively (right panel). Bound Nup88 was visualized by Western blot analysis with anti-Nup88 antibody. No binding of GST to the lamin tail domains was observed by immunoblotting with an anti-GST antibody.
	FIGURE 3:. Domain topology of endogenous and ectopically expressed Nup88 within the NPC. Nuclei were isolated manually and labeled with antibodies directly conjugated to 8-nm colloidal gold. (A) Schematic representation of Nup88 domain organization and antibody epitope. (B and D) Nup88 localizes to both the cytoplasmic and the nuclear side of the NPC with a polyclonal Nup88 antibody directed against the N terminus of Nup88 and a mAb against its central region. Shown are selected examples of gold-labeled NPCs in cross-section. c, cytoplasm; n, nucleus. (C and E) Quantitative analysis of the gold particle distribution associated with the NPC using an antibody against the N terminus of Nup88 and the central region. Sixty (N terminus) and 70 (center) gold particles were scored. (C) Quantitative analysis of the gold particle distribution associated with the NPC using an antibody against the N-terminal GFP-tag of Nup88. Fifty-five gold particles were scored. (F) Nup88 is found mainly on the cytoplasmic side of the NPC with a monoclonal Nup88 antibody directed against an epitope in the C terminus of Nup88. Shown are selected examples of gold-labeled NPCs in cross-section. c, cytoplasm; n, nucleus. (G) Left: Quantitative analysis of the gold particle distribution associated with the NPC using antibodies against the Nup88 C terminus. Fifty-seven gold particles were scored. Right: Quantitative analysis of the gold particle distribution with anti-myc antibodies. Thirty-eight gold particles were scored. Scale bars, 100 nm.
	FIGURE 4:. Expression of GFP-lamin A is masking an antibody epitope within the N terminus of Nup88. (A) HEK cells transfected with GFP-lamin A (d–f, g–i, and j–l) were prepared for immunofluorescence 24 h after transfection using mAbs directed against residues 314–425 of Nup88 (e and f). Cells transfected with GFP-lamin A showed a decreased signal for Nup88 staining as compared with untransfected control cells (a–c). The masking can be circumvented by the use of a polyclonal antibody directed against residues 27–45 in the N terminus of Nup88 (h and i) or methanol fixation in combination with the monoclonal Nup88 antibody against residues 314–425 (k and l). Scale bars, 10 μm. (B) Protein levels of Nup88 were analyzed in HEK cells transfected with GFP-lamin A or mock transfected control cells. No changes in protein levels of Nup88 were observed when compared with β-tubulin.
	FIGURE 5:. EDMD-associated lamin A(R453W) and FPLD-associated lamin A(R482W) mutants do not interact with Nup88. HEK cells transfected with GFP-lamin A(R453W) (d–f), GFP-lamin A(R482W) (g–i), GFP-lamin AΔ50 (j–l), or wild-type GFP-lamin A (a–c) were prepared for immunofluorescence 24 h posttransfection using mAbs directed against residues 314–425. Epitope masking was observed in cells transfected with wild-type GFP-lamin A (b) and GFP-lamin AΔ50 (k), respectively, but not in cells expressing the GFP-tagged lamin A(R453W) (e) and lamin A(R482W) (h) mutants.

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