FIGURE 2.

A, RNAi-mediated depletion of Axin leads to loss of K63-polyubiquitin-modified APC. HEK293 cells were transfected with siRNA for 72 h as indicated, followed by treatment with 10 μm MG132 for 4 h. Lysates were immunoprecipitated with K63 polyubiquitin linkage-specific antibodies, and analyzed by immunoblot as indicated. B and C, Wnt3a-induced loss of APC K63-adducts requires APC-Axin dissociation. HEK293 and HEK293-Myc-Axin1 cells were treated with 10 μm MG132 together with vehicle (−) or Wnt3a (200 ng/ml) for 2 h. Lysates were immunoprecipitated with K63 polyubiquitin linkage-specific antibodies (B) or anti-APC (C) and analyzed by immunoblot as indicated.