Figure 4. Biochemical properties of Nup188 and Nup192.

(A and B) Binding of MtNup188 and MtNup192 to various FG-coated beads monitored by in vitro pulldown assays. (A) Standard volumes of the soluble protein mixtures (pulldown input), separate components of the mixtures (protein alone, bacterial extract alone) and the 1M NaCl pulldown eluates form various FG-coated beads (1M NaCl pulldown elates) were precipitated by methanol-chloroform and re-solubilized in a standard volume of SDS-sample buffer. The samples were separated by SDS-PAGE and the gels were stained with SYPRO-Ruby. (B) The diagram representing relative enrichments of ScKap95, MtNup188, MtNup192 and 3xGFP in various FG-pulldown eluates, as compared to bulk bacterial extract proteins. The enrichment values (ratios between the eluted and input protein amounts) were computed using protein band intensities from the SYPRO Ruby stained gels shown in (A). (C) HsRanQ69L alone or an equimolar mixture of HsRanQ69L with ScKap95 were subjected to gel filtration using a Superdex 200 column, and the eluted fractions were separated by SDS-PAGE. Ran was visualized by Western blotting against the ZZ-tag, and ScKap95 was visualized by SYPRO Ruby staining. (D) Superdex 200 elution profiles of ZZ-HsRanQ69L (ZZ-tag Western blot) with or without the addition of MtNup188 or MtNup192 (SYPRO Ruby) analyzed as described in (C).

DOI: http://dx.doi.org/10.7554/eLife.00745.007

Figure 4—figure supplement 1. Characterization of the FG-repeat interactions in pulldown assays.

(A) The specificity of the FG-pulldown assay conditions were examined by subjecting bacterial extract proteins alone to pulldown assays with various FG- coated beads. Bound proteins were eluted with 1M NaCl sufficient to disrupt the expected transient interactions (left panel), followed by SDS sample buffer (right panel). The input bacterial extract sample and 1M NaCl eluates were precipitated by methanol-chloroform and re-solubilized in SDS-sample buffer prior to separation by SDS-PAGE. The gels were stained with SYPRO Ruby. (B) Proteins resistant to 1M NaCl elution in the experiment described in Figure 4A were stripped-off by SDS-sample buffer and separated by SDS-PAGE followed by SYPRO Ruby gel staining. The ‘protein sample’ lanes are the same as ‘protein alone’ in Figure 4A, and are included as references for the protein amounts remaining after 1 M NaCl elution.

DOI: http://dx.doi.org/10.7554/eLife.00745.008