Ab16898

Total protein was extracted from tissue samples using RIPA lysis buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protease inhibitors and phosphatase inhibitors (1:100) were added during protein extraction (MedChemExpress), and a Pierce BCA Protein Analysis Kit (Thermo Fisher Scientific) was used to measure protein concentrations. Protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk and incubated with the respective primary antibodies overnight at 4°C. The samples were incubated with horseradish peroxidase‐conjugated secondary antibodies (1:5000 dilution; Cell Signaling Technology) for 1 h at room temperature, and an iBright FL1500 imaging system (Invitrogen) and Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Invitrogen) were used to detect and analyse protein expression levels. Antibody information was provided for anti‐CD44 (60224‐1‐Ig; Proteintech), anti‐MYCN (ab16898; Abcam), and rabbit anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (WB: 1/1000; Cell Signaling Technology).