Western blot analysis of Wnt signaling genes. Western blot analyses were completed to examine the effects of deguelin on proteins of interest. To account for the difference in protein loading during the experiment, the percentage of regulation was calculated after the intensity of each band was adjusted according to its respective actin band intensity using the UnScan-It gel program (Silk Scientific, Inc.). A, the expression levels of total GSK-3β and GSK-3β Ser9, the phosphorylated form that represents the inactivated form of GSK3β. These studies show that deguelin (24-h treatment) increases expression levels of total GSK-3β protein while decreasing the levels of GSK-3β Ser9. Results also show downregulation of cyclin D1 after 24 h of deguelin treatment at the concentrations studied (0.250-5.0 μmol/L). B, the effect of deguelin treatment on total β-catenin protein levels was examined following 12 and 24 h posttreatment. We found that 24 h of treatment significantly reduces the expression levels of β-catenin.
