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. 2012 Jul 3;287(34):28552–28563. doi: 10.1074/jbc.M112.387878

FIGURE 5.

FIGURE 5.

A, ectopic expression of β-catenin increased the pool of K63-polyubiquitin-modified APC in HCT116 and HEK293 cells. Subconfluent cells were transfected with 2 μg of empty vector (−) or the indicated amounts of Myc-β-catenin expression vector for 48 h, followed by treatment with 10 μm MG132 for 4 h. Lysates were immunoprecipitated with K63 polyubiquitin linkage-specific antibodies or anti-E25 control, and analyzed by immunoblot as indicated. In HEK293 cells transfected with 2 μg of Myc-β-catenin expression vector, total APC levels were significantly reduced for reasons that are unclear, but this likely explains the reduced intensity of the ubiquitylated APC smear (lane 7). B, ectopic expression of wild-type (WT), but not β-catenin-ΔS45, increased the pool of K63-polyubiquitin-modified APC in HEK293 cells. Subconfluent cells were transfected the indicated amounts of empty vector, Myc-β-catenin-WT or FLAG-β-catenin-ΔS45 expression vectors for 24 h, followed by treatment with 10 μm MG132 for 4 h. Lysates were immunoprecipitated with K63 polyubiquitin linkage-specific antibodies or anti-E25 control, and analyzed by immunoblot as indicated.