FIGURE 7.
Nuclear activities of Axin-bound GSK3β and CKIα were increased in cells expressing Sox9. A, CHO cells were transiently transfected with β-CATENIN-HA and Sox9 mutants. β-Catenin degradation was inhibited by MG132. Cells were fractionated and nuclear extracts were analyzed by Western blot. The total amount of β-catenin was detected with anti-HA antibody. The membrane was stripped and re-probed with phospho-β-catenin-specific antibodies (Ser45 and Ser33/37). Lamins A/C and tubulin were used as markers for nuclear and cytoplasmic fractions, respectively. B and C, CHO cells were transiently transfected with Axin-myc, GSK3β-HA, and ckIα-HA, fractionated, and Axin-Myc was immunoprecipitated from the nuclear (B) and cytoplasmic (C) fractions. Equal amounts of immunoprecipitated Axin was loaded on the gel and analyzed with anti-HA antibody. The same membrane was also probed with anti-Myc antibody to confirm that an equal amount of Axin was loaded. D and E, equal amounts of GSK3β-HA or CKIα-HA bound to the immunoprecipitated Axin were subjected to in vitro kinase assays using phosphoglycogen synthase peptide-2 (P-GSP-2) as substrate for GSK3β and myelin basic protein for CKIα in the nuclear (D) or cytoplasmic (E) Axin immunocomplexes.