Anti cd133

For Western blot analysis, samples of the A549 cells induced by the small molecule chemical compounds were harvested, lysed in 200 μL RIPA lysis buffer (P0013B Beyotime, Shanghai, China), and centrifuged at 12000 rpm for 3 min. The cell lysate protein was quantified using the BCA Protein Assay Kit (Thermo Fisher). Then 15 or 20 μg of total protein sample were resolved on a 10% acrylamide gel for SDS-polyacrylamide gel electrophoresis and electrotransferred onto a PVDF membrane (Millipore, Darmstadt, Germany) at 150 mA for 120 min (Bio-Rad). Anti-CD133 (1:1000, GeneTex, Irvine, CA, USA), anti-vimentin (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (1:1000, Cell Signaling Technology), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-NOTCH1 (1:1000, Cell Signaling Technology), anti-NOTCH2 (1:1000, Cell Signaling Technology), anti-NOTCH3 (1:1000, Cell Signaling Technology), anti-HES1 (1:1000, Abcam, Cambridge, UK), GAPDH antibody (1:10000, Abcam), and horse radish peroxidase-conjugated goat-anti-rabbit IgG (1:1000, Cell Signaling Technology) antibodies were used. Pierce ECL liquid (Thermo Fisher) was used for exposure, which was performed using a gel imaging system (Bio-Rad).

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.