Anti fbxw7

Cells were washed twice with Hanks’ balanced salt solution and lysed in RIPA lysis buffer (50 mM Tirs-Cl, pH 7.4, 120 mM NaCl, 1% NP-40, 0.2% SDS, 1 mM EDTA and complete protease inhibitor), and centrifuged for 20 min at 13,000g, 4 °C. The protein concentrations were subsequently determined using a BCA Protein Assay Kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples (20 µg) were denatured with 4× loading buffer (TAKARA) at 95 °C for 5 min. The polyvinylidene difluoride (PVDF; Life Technologies) membrane was blocked with phosphate-buffered saline (PBS) supplemented with 5% non-fat milk. Equal quantities of protein were subjected to SDS-PAGE and gels were transferred onto PVDF membranes. The PVDF membrane was then incubated with the following anti-bodies: Anti-FBXW7 (1:500; Abcam) and anti-β-actin (1:2000; Cell Signaling Technology), at 4 °C overnight. The PVDF membrane was washed three times with PBS containing 5% Tween (Sigma-Aldrich), and incubated with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody at room temperature for 2 h. An ECL kit was used to visualize the protein bands according to the manufacturer’s instructions. The relative protein expression levels were analyzed using Image-ProPlus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The protein concentration was measured using a bicinchoninic acid (BCA) assay (#23225; Pierce, USA) according to the manufacturer’s instructions. The primary antibodies used in this study are as followed: anti-KDM5B (1:500, Cell Signaling Technology, #15327), anti-H3K4me3 (1:1000, Cell Signaling Technology, #9751), anti-H3 (1:1000, Cell Signaling Technology, #4499), anti-CCNE1 (1:1000, Cell Signaling Technology, #20808), anti-CCNE2 (1:1000, ProteinTech, 11935-1-AP), anti-CDK2 (1:1000, ProteinTech, 10122-1-AP), anti-FLAG (1:1000, Cell Signaling Technology, #8146), anti-FBXW7 (1:1000, Abcam, ab109617), anti-FLI1 (1:1000, Abcam, ab133485), and β-actin (1:5000, Cell Signaling Technology, #4970). The secondary antibodies are goat anti‐rabbit immunoglobulin G (1:2000, Abcam, ab7090) and goat anti‐mouse immunoglobulin G (1:2000, Abcam, ab97040). The unprocessed western blot images were shown in Supplementary Fig. S2.

Western blots (Wb) were performed as previously described [8 (link)]. The primary antibodies used were as follows: Anti-GAPDH (Cat. No. ab181602, Abcam, Cambridge, UK), anti-FBXW7 (Cat. No. ab109617, Abcam, Cambridge, UK), anti-beta-actin (Cat. No. ab8226, Abcam), anti-AR (Cat.no. ab133273) and anti-GATA2 (Cat. No. sc-515178, Santa Cruz Biotechnology, Dallas, TX, USA). The horseradish peroxidase (HRP)-conjugated secondary antibodies used were as follows: Anti-rabbit (Cat. No. NA934, Amersham, Buckinghamshire, UK), anti-mouse (Cat. No. 170-501, Bio-RAD, Hercules, CA, USA) and anti-goat (Cat. No. P0160, DAKO, Glostrup, Denmark).