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Supplementary Figure1 |
Specific competition of NES protein export by PKI-NES conjugates. A mixture of in vitro translated, radiolabeled GST-NES, GST-GST and importin a was injected into the oocyte nuclei. Where indicated, PKI-NES wild-type or mutant peptides conjugated to BSA were coinjected. After 2 hours, protein was extracted from either total oocytes (T), or, after dissection of the oocytes, from the cytoplasmic (C) and nuclear fractions (N). Proteins were separated by 10% SDS-PAGE and detected by autoradiography.
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Supplementary Figure 2 |
6His-hNMD3 (A) or 2z-NMD3 (B) cannot recruit CRM1 into a stable complex with cytoplasmic 60S subunits. Twenty-five pmol 60S ribosomal subunits were incubated with purified factors as indicated on the top of each panel [20 pmol 6His-hNMD3 or 2z-hNMD3, 10 pmol CRM1 and 75 pmol RanQ69L(GTP)]. Then, ribosomal subunits and associated factors were separated from unbound material by ultracentrifugation. Proteins from both fractions [supernatant (S) and pellet (P)] were precipitated and analyzed by 10% SDS-PAGE followed by Coomassie blue staining. Load of proteins in the unbound fractions equals the load of the ribosomal pellet fractions.
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