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. 2010 Jan 11;285(11):8472–8480. doi: 10.1074/jbc.M109.069450

FIGURE 3.

FIGURE 3.

A, Western blot analysis showing the kinetics of GSK3β phosphorylation of β-catenin under control conditions (PCMV) or after Peg3/Pw1 protein overexpression. LiCl (30 mm) was used to inhibit GSK3β. Phosphorylated β-catenin was detected using a phosphospecific antibody. MG132 (25 μm) was added to inhibit β-catenin degradation. B, Western blot illustrating the effect of Peg3/Pw1 shRNA on expression of FLAG-tagged wild-type (WT) or mutant (MT) β-catenin. C, Western blot illustrating the effect of proteasome inhibition by MG132 (25 μm) on Peg3/Pw1-mediated degradation of β-catenin. D, Western blot illustrating the effect of DNA damage induced by camptothecin (CPT; 50 μm) on β-catenin protein expression in HeLa, 293T, U251, and D566 cells. Cont, control. E, Western blot illustrating the effect of DNA damage induced by camptothecin (50 μm) on β-catenin expression after Peg3/Pw1 knockdown in D566 cells. F, Western blot illustrating the effect of Peg3/Pw1, Peg3/Pw1 shRNA, Siah1, and Siah1 shRNA on β-catenin expression in 293T cells. Note that the combination of Siah1 and Peg3/Pw1 decreased β-catenin to a greater extent compared with Peg3/Pw1 after Siah1 knockdown. G, anti-HA Western blot (WB) of Myc-Siah1 immunoprecipitates (IP) after cotransfection of Myc-Siah1 and HA-Peg3/Pw1 (second lane), HA-Peg3SCAN (third lane), and HA-Peg3ZF (fourth lane) in 293T cells. H, Tcf/Lef luciferase reporter assay in 293T cells after overexpression of the human Peg3/Pw1, Peg3/Pw1 shRNA, and/or Siah1 vector. Data shown are means ± S.E.