Ab10540

Embryos were dissected from pregnant females at gestational stage 8.5~9.5 and the deciduas were torn off. Total proteins were extracted with Mammalian Protein Extraction Reagent (M-PER; Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (P8340, Sigma-Aldrich). Protein concentrations were determined with a Pierce BCA protein assay kit (Thermo Scientific). Appropriate amounts of the extracts were fractionated by SDS-PAGE, electrotransferred to PVDF membranes, and detected with antibodies against CUL4B (HPA011880, Sigma-Aldrich), β-catenin (14–6765, ebioscience, San Diego, CA, USA), phospho-β-catenin (S33/S37/T41) (9561, Cell Signaling Technology, Danvers, MA, USA), cyclin D1 (ab10540, Abcam, Cambridge, MA, USA), cyclin D2 (ab3087, Abcam), cyclin D3 (ab28283, Abcam), cyclin E (630701, Biolegend, San Diego, CA, USA), and β-actin (A5060, Sigma-Aldrich). Signals from the reaction with anti-mouse IgG and anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (AP124P and AP132P, Millipore) were developed with the Immobilon Western Chemiluminescent HRP substrate (WBKLSO500, Millipore). The signals were semiquantified using Multi Gauge V3.0 software (Fujfilm) and normalized against that of β-actin.

In order to analyze the expression of Ki-67 and Cyclin D1, LNCaP cells were treated for 48 h with complex 1 at a concentration of 6.5 µM (determined from the IC50 value). After treatment, cells were washed with PBS (Phosphate-Buffered Saline), and permeabilized by BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Pharmingen, San Jose, CA, USA). Subsequently, LNCaP cells were stained with anti-Ki67 (1:100; RM360, Sigma) and anti-Cyclin D1 (1:100; ab10540, Abcam, Cambridge, UK) for 1 h at room temperature. Anti-rabbit IgG-FITC (1:200, 656111, Thermo Fisher Scientific) and anti-mouse IgG-Atto 647N (1:50, 50185, Sigma Aldrich) were used as a secondary antibody, respectively. Cell staining was analyzed using flow cytometry (Accuri C6, BD Pharmingen).