Figure 2.

Nup62 interacts with TIP60 via the coiled-coil domain. (A) The schematic representation of different Nup62 truncations and deletion mutant generated to pin down the TIP60-binding activity. FL, full-length, aa 1–522; ΔCC, coiled-coil domain deleted mutant, Δaa 328–458; NT, N-terminal, aa 1–328; CT, C-terminal, aa 329–522. (B) Immunofluorescence images for localization of transiently expressed GFP-Nup62-FL, NT, and CT in interphase and mitotic Hela cells. Cells were synchronized with 2 mM thymidine for 16 h in G1/S before fixing with 4% formaldehyde or released from thymidine for 8 h to observe the protein localization in mitotic cells. Pericentrin was stained to indicate centrosome. Scale bar, 5 µm. (C) Temporal expression profile of Nup62 during cell cycle. Western blotting analyses of Nup62 in G1/S-synchronized HeLa cells. Note that Nup62 level is comparatively stable, unlike cyclin B1 levels that change from interphase to mitosis. GAPDH served as a loading control. WCL, whole-cell lysate. (D) In vitro pull-down assay between GST-TIP60 and MBP-Nup62 FL and deletion mutants. GST-TIP60-bound agarose beads were used as affinity matrices to absorb the MBP-Nup62 FL or deletion mutants purified from bacteria. The affinity matrices were subjected to western blotting analysis after extensive washes with the indicated antibodies (upper panel) or CBB staining (lower panel). The bound or absorbed fraction of MBP-Nup62-CT with GST-TIP60 is indicated with an arrow.