FIG. 3.

Keap1 is a NES-containing nuclear-cytoplasmic shuttling protein. (A to C) Treatment of HeLa cells with LMB induces nuclear translocation of the ectopically produced full-length Keap1 (A), of the EGFP-Keap1 fusion protein (B), and of endogenous Keap1 (C). Confocal images were obtained by using anti-Keap1 2H5 monoclonal antibody (A and C) or intrinsic fluorescence of EGFP (B). (D) Recognition of endogenous Keap1 in HeLa cells is prevented by preincubation of the anti-Keap1 2H5 monoclonal antibody with recombinant GST-Keap1(308-624) (+GST-Keap1 panel) but not with GST alone (+GST panel). Images were acquired with identical confocal microscope settings. (E) Schematic representation of the Keap1 mutants used for the identification of the NES of human Keap1. DGR, diglycine repeat; wt, wild type. The indicated proteins were fused to the C terminus of EGFP. (F) Western blot analysis of the production of the EGFP-fused Keap1 mutants shown in panel E in transfected HeLa cells. Anti-EGFP antibody was used for detection of the respective proteins. (G) Confocal images showing intracellular localization of the Keap1 mutants fused to EGFP in HeLa cells either untreated or treated with LMB for 4 h. The results presented are also summarized in panel E, right column. Bars, 10 μm.