Figure 2.

Immunodetection of β-catenin using phosphospecific antibodies. The cell lysates described in Figure 1b were probed with the following antibodies: (a) antiphospho-β-catenin 33/37/41, (b) antiphospho-β-catenin 41/45 and (c) α-ABC (α-active β-catenin, for β-catenin unphosphorylated at Ser45 and Thr41). (d) An in vitro kinase assay was performed with purified GST-tagged rAxin, GSK-3β and WT or mutant β-catenins, in the presence and absence of ATP. Reaction products were analysed by SDS–PAGE with immunodetection using anti-pβ-catenin-33/37/41 antibody. Equal protein loading was confirmed by silver staining (bottom); the last three lanes, respectively, were controls lacking β-catenin but containing GSK-3β plus rAxin, GSK-3β alone or rAxin alone