Figure 1. In vitro reconstitution of Axin-dependent and CK1 priming-dependent β-catenin phosphorylation by GSK3.A. Recombinant GST-β-catenin, Flag-CK1, MBP-Axin, and His-GSK3 proteins were expressed in bacteria or insect cells and purified by glutathione agarose, anti-Flag M2 agarose, amylose resin, or Ni-NTA resin, respectively. In the case of β-catenin, GST was cleaved via thrombin and purified away from β-catenin. * indicates each recombinant protein. B. β-catenin phosphorylation by CK1 was reconstituted in vitro using purified proteins. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser45 β-catenin antibody. C. Axin-dependent phosphorylation by GSK3 was reconstituted in vitro using purified proteins. For Axin-dependent β-catenin phosphorylation in this and other figures, 0.43 µM of GSK3, 0.54 µM of CK1α, 0.21 µM of Axin, and 0.73 µM of β-catenin were used in each assay. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser45 β-catenin antibody and an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody.
Image published in: Wu G et al. (2009)
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