(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).