Fig. 5. The microtubule-binding activity, but not the DNA binding activity of Kid is inhibited by the importin /ß complex. (A) The amino-terminal domain (KNT) and the carboxy-terminal domain (KCT) of Kid and BSA were immobilized onto Affiprep 10 beads, incubated with mitotic HeLa cell extract then re-isolated and analyzed by SDS-PAGE and immunoblotting to detect importin ß. (B) GST-KCT was incubated with DNA cellulose at 4°C for 1 hour and then successively washed with buffers containing 50 mM NaCl (50), 300 mM NaCl (300) and 500 mM NaCl (500). The eluates were analyzed by SDS-PAGE and Coomassie Blue staining. L, load. (C) GST-KCT was incubated with DNA cellulose in the presence or absence of GST-RanL43E and the importin /ß complex. The DNA cellulose was washed with 50 mM NaCl followed by 500 mM NaCl. The initial amount of KCT (L) incubated with the DNA cellulose was compared to that eluted with 500 mM NaCl (E). (D) GST-KNT was incubated with Taxol-stabilized microtubules in the presence or absence of GST-RanL43E and the importin /ß complex. The microtubules were re-isolated by centrifugation and the subsequent pellet (P) and supernatant (S) fractions analyzed by SDS-PAGE and immunoblotting to detect GST.