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Fig. 2. A23187 promotes ß-catenin degradation through GSK-3ß-independent and ß-TrCP-dependent mechanism. (A) HEK293 reporter cells were incubated with A23187 (0.625, 1.25, 2.5 and 5 µM) in the presence of 20 mM LiCl. After 15 hours, luciferase activity was determined. (B) Cytosolic proteins were prepared from HEK293 reporter cells treated with either vehicle (DMSO) or A23187 (2.5 µM) in the presence of 20 mM LiCl for 15 hours and then subjected to western blotting with ß-catenin antibody. The blots were reprobed with anti-actin antibody as a loading control. (C) HEK293 cells were co-transfected with the indicated plasmids and then incubated with either the vehicle (DMSO) or A23187 (2.5 µM) for 15 hours. Cytosolic proteins were subjected to western blotting with ß-catenin antibody. The blots were reprobed with anti-GFP antibody as a transfection control. (D) HEK293 reporter cells were co-transfected with the indicated plasmids and then incubated with either the vehicle (DMSO) or A23187 (2.5 µM) for 15 hours and then luciferase activity was measured. In A and D, the results are the average of three experiments, and the bars indicated standard deviation.
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