CGK062 promotes PKCα-mediated β-catenin phosphorylation/degradation.

<p>(A) Cytosolic and membrane fractions were prepared from HEK293 cells treated with vehicle (DMSO) or CGK062 (25 and 50 µM) in the presence or absence of Wnt3a-CM for Western blotting with anti-PKCα antibody. The blots were reprobed with anti-tubulin or anti-MDR antibodies as fraction controls. (B) Indicated peptide was incubated with purified PKCα and CGK062 (12.5 and 25 µM) or BIM (0.5 µM). The amounts of ATP in the reaction were detected by Kinase-Glo™ Luminescent Assay kit (Promega). The results are the average of three experiments, and the bars indicate standard deviations. *, <i>P</i><0.05, compared with the vehicle control group. (C) Cytosolic proteins were prepared from HEK293 reporter cells treated with CGK062 (25 µM) or BIM (10 µM) in the absence or presence of Wnt3a-CM for Western blotting with anti-β-catenin antibody. (D) HEK293 reporter cells were transfected with negative control (NC) siRNA (40 nM) or PKCα siRNA (40 nM), and then incubated with CGK062 (25 µM) for 15 h in the absence or presence of Wnt3a-CM. Cell lysates were subjected to Western blot analysis with anti-PKCα and anti-β-catenin antibodies. In (C) and (D), the blots were reprobed with anti-actin antibody to confirm equal loading. (E) GST-β-catenin (100 ng) was incubated with purified PKCα and CGK062 (12.5 and 25 µM) or BIM (0.5 µM). The samples were analyzed by Western blotting with anti-phospho-p33/37-β-catenin antibody. (F) HEK293 reporter cells were incubated with CGK062 (25 µM) and BIM (10 µM) for 15 h in the absence or presence of Wnt3a-CM. Cytosolic fractions were prepared and subjected to Western blot analysis with anti-phospho-p33/37-β-catenin or anti-β-catenin antibody. (G) HEK293 reporter cells were transfected with negative control (NC) siRNA (40 nM) or PKCα siRNA (40 nM), and then incubated with CGK062 (25 µM) for 15 h in the absence or presence of Wnt3a-CM. Cell lysates were subjected to Western blot analysis with anti-β-catenin and anti-p33/37-β-catenin antibodies. In (F) and (G), the same amount of β-catenin was loaded in each lane.