FIGURE 2:

The NLSs of hsNXF1 for Impβ, Kapβ2, Imp4, Imp11, and Impα are all located in the N-terminal tail (hsNXF1-N). (A) Summary of the pull-down binding assays of hsNXF1 domains with Impβ, Kapβ2, Imp4, Imp11, and Impα (data shown in Supplemental Figure S1). The number of plus signs indicates the relative binding strength, and the minus sign indicates no significant binding. (B) The sequence of hsNXF1-N. The two NLS epitopes identified by alanine scanning mutagenesis and ITC (Supplemental Figures S3–S7 and Table 1) are underlined. (C) Alanine mutations at both NLS epitopes of hsNXF1 eliminated binding to Impβ, Kapβ2, Imp4, Imp11, and Impα. Immobilized GST-karyopherins were incubated with MBP-hsNXF1-N or mutant MBP-hsNXF1-N(21-30, 71-75/A). Bound proteins were visualized by SDS–PAGE and Coomassie staining. GST-Impα* refers to Impα without its N-terminal IBB domain (residues 75–529).