Apigenin-induced proteasome-dependent degradation of Hsp90/Cdc37 client proteins are correlated with inhibition of CK2. (A) HeLa cells were treated with the indicated concentrations of apigenin or TBB for 24 h. The protein levels of CK2α, RIP1, Raf-1 and Cdk4 were detected by western blot analysis. (B) HeLa cells were transiently transfected with control siRNA or CK2α siRNA for 48 h. Whole-cell lysates were analyzed by western blotting using the indicated antibodies. (C) Next, siRNA was introduced intoU266 and RPMI 8226 cells using electroporation. After 48 h, the cells were harvested to detect the protein levels by western blot. β-actin, as well as α-tubulin, served as loading control. (D) U266 cells were pretreated with MG132 (1 μM) for 1 h, and the cells were subsequently treated with apigenin (90 μM) for an additional 12 h. Whole-cell lysates were subjected to western blot analysis using antibodies against Raf-1, Src, Cdk4 and β-actin. (E) and (F) U266 cells were incubated with or without the Hsp90 inhibitor GA (0.2 μM) or SAHA (1 μM) for 24 h in the presence or absence of 30 μM apigenin. Whole-cell lysates were subjected to western blotting to determine the levels of Raf-1, Src, Cdk4 and β-actin. The bar graphs on the right show the percentage of intensities of the protein band from each treatment relative to the controls, which were defined as 100%. Values represent the means ± SD. *Significant difference from the three groups was designed by ANOVA, **p < 0.01.