Fig. 6
Evaluation of the inhibitor-dependent mechanism for ERα degradation. A In vitro ERα competitive binding assays for MK8776 (MK), CCT241533 (CCT), VE822 (VE), KU60019 (KU), AZD7762 (AZD) prexasertib (Prexa), GDC-0575 (GDC), and 17β-estradiol (E2) were performed at different doses of the compounds and using a florescent E2 as the tracer. Relative inhibitor concentration 50 (IC50, i.e., Kd) is given in the graph. The experiment was performed twice in quintuplicate. Western blot (B-C) and relative densitometric analysis (D) of ERα levels in MCF-7 (B) and Y537S (C) cells pre-treated with cycloheximide (CHX) at the indicated doses for 6 h and then treated with AZD7762 at the indicated dose for 24 h. The loading control was done by evaluating vinculin expression in the same filter. The number of replicates is given as solid dots in the bar graph. Significant differences with respect to CTR sample are calculated by Student t-test and indicated by **** (p-value < 0.0001). Significant differences with respect to the CHX sample are calculated by Student t-test and indicated by ° (p-value < 0.05), and °°°° (p-value < 0.0001)