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. 2000 Oct 16;151(2):321–332. doi: 10.1083/jcb.151.2.321

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© 2000 The Rockefeller University Press

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D23A p10 is a dominant-negative inhibitor of the nuclear import of TRITC–BSA–NLS in vitro. (A) wt p10 or D23A p10 (both at 3.0 μM dimer concentration) were added to an import solution consisting of 10 μg/ml TRITC–BSA–NLS, 10 mg/ml Xenopus ovarian cytosol, 0.5 mM GTP, and 1 mM ATP plus a regenerating system. These mixtures were incubated with permeabilized BRL cells for 20 min at room temperature before washing and fixation. (B) The import assay was done as described in A, except purified transport factors were used to support import, rather than the cytosol, and the reaction was for 15 min. The import reactions contained 10 μg/ml TRITC–BSA–NLS, 0.5 μM Kap-α2, 0.25 μM Kap-β1, 2 μM RanGDP, 0.5 mM GTP, and 2 mg/ml BSA in addition to the indicated concentration of wt or D23A p10 dimer.

D23A p10 is a dominant-negative inhibitor of the nuclear import of TRITC–BSA–NLS in vitro. (A) wt p10 or D23A p10 (both at 3.0 μM dimer concentration) were added to an import solution consisting of 10 μg/ml TRITC–BSA–NLS, 10 mg/ml Xenopus ovarian cytosol, 0.5 mM GTP, and 1 mM ATP plus a regenerating system. These mixtures were incubated with permeabilized BRL cells for 20 min at room temperature before washing and fixation. (B) The import assay was done as described in A, except purified transport factors were used to support import, rather than the cytosol, and the reaction was for 15 min. The import reactions contained 10 μg/ml TRITC–BSA–NLS, 0.5 μM Kap-α2, 0.25 μM Kap-β1, 2 μM RanGDP, 0.5 mM GTP, and 2 mg/ml BSA in addition to the indicated concentration of wt or D23A p10 dimer.