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. 2011 Aug 8;286(39):33921–33930. doi: 10.1074/jbc.M111.272906

FIGURE 6.

FIGURE 6.

Importin β cooperates with Ran to promote NHK ubiquitination. A, Ran is required for NHK ubiquitination in vitro. Ran in the cytosol was depleted (−Ran cyt) by incubating the cytosol with Ni-NTA-agarose prebound with His-tagged importin β-N603 at 4 °C for 2 h. Mock control was only incubated with Ni-NTA-agarose. His-tagged recombinant Ran is ∼1 kDa larger than endogenous Ran. B, quantification of NHK ubiquitination in A. The data represent the means ± S.E. of three independent experiments. The relative intensity of the polyubiquitin smear (Ubn-NHK) in the reaction with mock cytosol for 1 h (lane 6) was set to 1. C, effects of N-terminal truncation mutants of importin β on NHK ubiquitination in vitro. The diagram represents recombinant proteins used in this experiment. 1 μg of each recombinant protein was used as indicated. D, quantification of NHK ubiquitination in C. The data represent the means ± S.E. of three independent experiments. The relative intensity of polyubiquitin smear (Ubn-NHK) in reaction with control cytosol for 1 h (lane 8) was set to 1. Ran binding was quantified from supplemental Fig. S6A. The relative band intensity of Ran bound to WT importin β was set to 1. Imp, importin.