Figure 4.

FG Nups within NPCs in purified nuclei are hypersensitive to protease digestion. (A) Purified nuclei (4 mg/ml) were digested with proteinase K (300 ng/ml) for the indicated times at 37°C. Aliquots were mixed with SDS buffer with 2 mM PMSF to stop the digestion. The degradation of specific proteins was quantified by using Western blots probed with specific antibodies and ¹²⁵I-Protein A. For each protein, the fraction of full-length protein remaining is shown. Each graph represents the average of three to six independent digestions; error bars represent the SEM. The top line in Mex67p represents the sum of full-length and clipped Mex67p (*); the bottom line represents full-length Mex67p. (B) Representative ¹²⁵I-Protein A Western blots used to generate graphs shown in A. Note that in the case of Mex67p, the full-length protein is clipped to yield two large, stable fragments (*). (C) Purified FG repeat regions of Nups (1 mg/ml) with and without stoichiometric amounts of Kap95p–Kap60p were incubated with proteinase K (100 ng/ml) for the indicated times at 37°C. Samples were processed as in A, resolved by SDS/PAGE, and visualized by Coomassie blue staining.