FIG. 2.
Subcellular localization of Axam. (A) Subcellular localization of Axam mutants. DLD-1 cells were stained with the anti-Axam antibody incubated with GST (a) or GST-Axam-(72-588) (b). The lysates (20 μg of protein) of DLD-1 cells were probed with the indicated anti-Axam antibodies (Ab) (c). SW480 cells expressing GFP-Axam mutants were stained with the anti-GFP antibody (d to h). (B) Nuclear accumulation ofAxam by leptomycin B treatment. After COS cells expressing GFP-Axam-(72-400) (a to c) or GFP-Axam-(381-588) (d and e) had been treated with (+; c and e) or without (−; a, b, and d) 20 ng of leptomycin B/ml for 30 min, the cells were visualized under a confocal laser scanning microscope. The nuclei in image a were visualized by DAPI (b). LMB, leptomycin B. Arrows indicate the cells expressing GFP-Axam-(72-400). (C) Colocalization of Axam and Axin. SW480 cells expressing Myc-rAxin alone (a), Myc-rAxin and GFP-Axam (b to d), Myc-rAxin and GFP-Axam-(72-400) (e to g), or Myc-rAxin and GFP-Axam-(381-588) (h to j) were stained with the anti-Myc antibody to detect Myc-rAxin (a, c, f, and i) and the anti-GFP antibody to detect GFP-Axam and its mutants (b, e, and h). The merged image shows colocalization of Myc-rAxin with GFP-Axam or GFP-Axam-(72-400) but not with GFP-Axam-(381-588) (d, g, and j).