Anti abcg2

An antibody against PAR1 was purchased from BECKMAN COULTER. Anti-E-cadherin, fibronectin, YAP1, phospho-YAP1 (pYAP1), Lats1 and phospo-Lats1 (pLats1) were purchased from Abcam. Anti-ABCG2, -MRP1, and -P-glycoprotein (P-gp) were purchased from GeneTex. Anti- ribophorin II (RPN2) was purchased from OriGene. Anti-GAPDH was from IMGENEX. The selective PAR1 agonist TFLLR-NH₂ and PAR1 antagonist SCH79797 was purchased from Tocris Bioscience. We used TFLLR-NH₂ (EC50: 1.9 μM) at the 20 μM, and SCH79797 (IC50: 70 nM) at the 70 nM. C3 transferase and Y27932 were purchased from Cytoskeleton. We used C3 at the 2 μg/ml and Y27632 at 10 μM. Small interfering RNA (siRNA) directed against PAR1 (5′-AAGGCUACUAUGCCUACUACU-3′) was synthesized by TOYOBO, and siRNA directed against YAP1 (5′-GGUCAGAGAUACUUCUUA-3′) and Snail (5′- CCACAGAAAUGGCCAUGGGAAGGCCUC-3′) were synthesized by Sigma. Control siRNA is a scrambled sequence with no homology in the human genome (Qiagen) is listed as follows: Scrambled, UUCUCCGAACGUGUCACGUdTdT.

Cells were collected by sample buffer and analyzed by electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF, Millipore) membrane and TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20) containing 5% nonfat milk was used for blocking. Anti-USP24 (Proteintech), anti-p300 (BD), anti-actin (Sigma-Aldrich), anti-ubiquitin (Santa Cruz), anti-P-gp (Genetex), anti-ABCG2 (Genetex), anti-MRP1 (Genetex), anti-MRP3 (Genetex), anti-Ezrin (Genetex), lamin A/C (Santa Cruz), anti-γ-H2Ax (abcam), anti-biotin (Genetex), anti-Flag (Genetex), anti-Rad51 (abcam), and anti-BRD7 (Sigma-Aldrich) were used for probing interested proteins. After incubated with primary antibodies, PVDF membranes were then incubated with secondary immunoglobulin antibodies linked with horse radish peroxidase (Millipore, 1:10,000). For detecting immunoprecipitated samples, light chain-specific secondary antibodies were used (Jackson ImmunoResearch, 1:10,000). ECL Western blotting detection system (Millipore) and ChemiDoc-it imager (UVP) were used for detecting signals.

The spheroids were formed by plating 1 × 10⁶ cells per well in serum-free media with supplementation of growth factors and treated with various concentrations of aqueous extractions of BJ (0, 5, 10, and 15 mg/mL) for 12 h. The harvested cell pellets were lysed and the protein concentrations determined using a Bio-Rad protein assay kit for (Hercules, CA, USA) before being resolved by electrophoresis and transferred to nitrocellulose membrane. The blots were blocked with 5% non-fat milk and incubated with 1:2000 dilutions of primary antibodies, including anti-pAkt (GTX128414); anti-PARP (GTX112864); anti-EGFR (GTX121919); anti-pEGFR (GTX61507), anti-ABCG2 (GTX100437), anti-Nanog (GTX100863), anti-CD133 (GTX100567), anti-Sox2 (GTX627405), and anti-ALDH1A1 (GTX123973), from GeneTex. Membranes were then incubated with 0.3 µg/mL of peroxidase-conjugate anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific) and detected with enhanced chemiluminescence substrate (Thermo Fisher Scientific). The loading control was incubated with anti-GAPDH antibody (GTX100118, GeneTex). The signals were visualized with enhanced LAS-4000 (FUJIFILM) apparatus and the band intensities of images analyzed using ImageJ software.