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Fig. 4. PKC is required for A23187-mediated degradation of ß-catenin. (A) A23187 increases the intracellular Ca²⁺ concentration. HEK293 reporter cells were incubated with vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM for 15 hours. After fixation, the cells were stained with Fura-2/AM as described in Materials and Methods and observed at 400x magnification. (B) HEK293 reporter cells were incubated with A23187 (2.5 µM) or CsA [2.5 (+), 5 (++) µM], KN-93 [2.5 (+), 5 (++) µM] and BIM [2.5 (+), 5 (++) µM] for 15 hours in the presence or absence of Wnt3a-CM, and the luciferase activity was determined. The results are shown as the average of three experiments; the bars indicate standard deviations. (C) Cytosolic proteins were prepared from HEK293 reporter cells treated with A23187 (2.5 µM) or BIM (5 µM) in the absence or presence of Wnt3a-CM for western blotting with anti-ß-catenin antibody. The blots were reprobed with anti-actin antibody as a loading control.

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