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. 2004 Apr 1;23(9):1934–1942. doi: 10.1038/sj.emboj.7600198

Figure 7.

Figure 7

14-3-3 masks phosphorylated-p27NLS from importin α binding. (A) Specific 14-3-3 isoforms bind to phosphorylated p27NLS. Recombinant HA-14-3-3 isoforms (β, ɛ, γ, σ, θ and ζ) were incubated with bBSA-NLS (lanes 1, 3, 5, 7, 9 and 11) or bBSA-pNLS (lanes 2, 4, 6, 8, 10 and 12). The bound proteins were eluted and analyzed by SDS–PAGE. (B) 14-3-3 competes with importin α for binding to Thr157-phosphorylated p27NLS. Importin α5 was incubated with bBSA-NLS (lanes 2–5) or bBSA-pNLS (lanes 7–10) in the presence of increasing amounts of HA-14-3-3γ (lanes 3–5 and 8–10). The amounts of added HA-14-3-3 are 2 (lanes 3 and 8), 6 (lanes 4 and 9) and 16 (lanes 5 and 10) molar excess of importin α5. (C) 14-3-3 inhibits the nuclear import of Thr157-phosphorylated p27NLS in vitro. Conjugates were subjected to the in vitro reconstituted transport assay using importin α5/β with Ran and NTF2 in the presence of increasing amounts of HA-14-3-3γ. The amounts of added HA-14-3-3 are 8 and 24 molar excess of importin α5.