Goat anti rabbit immunoglobulin g

Cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The total protein concentrations of the lysates were determined using the Bio-Rad protein assay kit. Equal amounts of protein were denatured with loading buffer (Beyotime) at 100°C for 10 min, then loaded in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis for electrophoresis and transferred to a methanol-activated polyvinylidene fluoride membrane (Millipore, United States). The membrane was blocked in tris-buffered saline containing 5% bovine serum albumin (MP Biomedical, United States) for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C. The primary antibodies included AKT2, CD133, DNMT3B, MYC, PIK3R1, RBL1 (1:1,000, Proteintech, United States), Bcl-2 (1:1,000, Affinity Biosciences, United States), and GAPDH (1:2,000, ZSGB-BIO, China). After washing with tris-buffered saline twice, the membrane was incubated with the appropriate horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. The secondary antibody conjugated with horseradish peroxidase is goat anti-rabbit immunoglobulin G (1:5,000, ZSGB-BIO). Immunoblots were visualized using enhanced chemiluminescence detection system according to the manufacturer’s protocol.