FIGURE 6.

Sox9 binds GSK3β and promotes its nuclear translocation. A, GSK3β-HA was co-immunoprecipitated (co-IPed) with Sox9-Flag when transiently co-expressed in CHO cells. B, CHO cells were transiently transfected with a vector alone or the indicated plasmids. Cells were fractionated and subcellular distribution of endogenous GSK3β was analyzed by Western blot by anti-GSK3 antibody. Sox9 and its mutants were detected by anti-FLAG antibody. Lamins A/C and tubulin served as markers for nuclear and cytoplasmic fractions, respectively. Sox9HMG, although at a lower level, was also detected in the nuclear fraction, possibly due to some contamination by the cytoplasmic fraction. C, CHO cells were transiently transfected with the indicated plasmids. GSK3 was immunoprecipitated using anti-HA antibody. Sox9 was detected with anti-FLAG antibody. D, CHO cells were transiently transfected with β-CATENIN-HA and the indicated plasmids and analyzed by Western blot. Tubulin served as loading control. E and F, NCI-H28 (E) or SNU475 (F) cells were infected with Sox9-adenovirus and localization of endogenous GSK3β (green) and Sox9 (red) were detected 24 h later. DAPI stained the nucleus (blue). G and H, NCI-H28 (G) or SNU475 (H) cells were transfected with βTrCP-myc and infected with Sox9-adenovirus when indicated. Localization of βTrCP-myc (green) and Sox9 (red) was detected by immunofluorescence. DAPI stained the nucleus. IB, immunoblot.