FIGURE 6.

Co-immunoprecipitation analysis of ILKAP-importin interactions. A, importin α and importin β were found to interact with the GST-ILKAP fusion proteins through a GST pulldown assay. The GST and GST fusion proteins expressed in E. coli were immobilized on glutathione-Sepharose beads. After extensive washing, the beads were incubated with equal amounts of protein extracts from untransfected A549 cells. The retained proteins were analyzed by immunoblotting with anti-GST, anti-importin α, or anti-importin β antibodies. B, the lysates of COS-7 cells expressing Myc-importin α proteins (importin α1, α3, and α5) and EGFP-ILKAP were immunoprecipitated with an anti-Myc antibody. Each precipitate sample was blotted with anti-Myc and anti-EGFP antibodies to detect importin α (Myc) and ILKAP (EGFP). Ctrl-IgG, mouse IgG and beads (negative control). C, the lysates of COS-7 cells expressing various EGFP-ILKAP constructs (residues 1–107, 1–200, 108–392, NΔ71–86, FΔ71–86, or full-length protein) and Myc-importin α1 were immunoprecipitated with an anti-EGFP antibody. Each precipitate sample was blotted with anti-Myc and anti-EGFP antibodies to detect both importin α1 (Myc) and ILKAP (EGFP).