Figure 3. The NES-like sequence is exposed only in SOD1 mutants but not in wt SOD1. ; (A) Structural localization of the NES-like peptide in wt SOD1. The X-ray crystallographic structure of wt SOD1 (PDB: 2c9v, only chain A of the dimer was shown) is analyzed in UCSF Chimera 1.8. Key residues in the NES-consensus sequence are labeled in red, whereas other residues are labeled in blue. Note that only Leu42 is exposed in the surface. Upper panel: ribbon structure; lower panel: surface structure. (B) GST pull-down. Immobilized GST or GST-tagged protein as indicated was incubated with recombinant CRM1 and RanGTP. (C) RanGTP-dependent interaction between GST-SOD1G93A and CRM1. Recombinant CRM1 was precipitated by immobilized GST, GST-SOD1wt or GST-SOD1G93A with or without RanGTP. (D) Native immunoprecipitation (IP). GFP-SOD1wt and GFP-SOD1G93A were each expressed in HEK293T cells. The cell lysates prepared under native condition were immunoprecipitated with either the preimmune serum (control) or antibodies against a peptide containing the NES-like sequence of SOD1 (α-NLP). (E) Native IP. HEK293T cells expressing different GFP-tagged SOD1 proteins were lysed under native condition and immunoprecipitated with α-NLP. WCL: whole cell lysates.

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