Fig. 6. GSK-3ß is inactivated in lens cells treated with Wnt CM. (A) Explants exposed to FGF2 for 1 hour were washed with 2 M NaCl, and cultured in control medium or Wnt3a CM for 5 days. Explants were immunolabeled by using anti-Ser9 phospo-GSK-3ß (a,b), or Hoechst 33258 to stain nuclei. Ser9 phospho-GSK-3ß accumulated in explants cultured with FGF2/Wnt CM (b), but not in explants cultured with FGF2/Control medium (a). (B) In vitro GSK-3b kinase assays were performed using a GSK-3 substrate and [-³²P]. Lysates containing 150 µg of protein prepared from explants cultured with FGF2/Control medium (F/C) or FGF2/Wnt CM (F/W) were immunoprecipitated with anti-GSK-3ß and analyzed using a GSK-3ß immune complex kinase assay. The activity of GSK-3ß was decreased in explants cultured with FGF2/Wnt CM compared with explants cultured with FGF2/Control medium. Values represent the mean±s.d. of three independent experiments and are expressed as a percentage of the activity in FGF2/Control explants.