Methanol activated polyvinylidene fluoride membrane

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.

Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The total protein concentrations of the lysates were determined using a protein assay kit (Bio-Rad, USA). Equal amounts of protein were denatured with loading buffer (Beyotime, Shanghai, China) at 100°C for 10 min, then loaded in 12% SDS-PAGE for electrophoresis, and transferred to a methanol-activated polyvinylidene fluoride membrane (Millipore, USA). The membrane was blocked in tris-buffered saline (TBS) containing 5% bovine serum albumin (MP Biomedical, USA) for 2h at room temperature. Primary antibodies were incubated overnight at 4°C. The primary antibodies include: RBL1 (1:1000, Proteintech, China), CCND1, CCNF (1:1000, Affinity Biosciences, USA) and GAPDH (1:1000, ZSGB-BIO, Beijing, China). After washing with TBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature. Secondary antibody conjugated with HRP is Goat-Anti Rabbit IgG (1:5000, ZSGB-BIO, Beijing, China). The relative protein level is quantified using Image J software.

Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). Lysate total protein concentrations were determined using a protein assay kit (Bio-Rad, USA). Equal amounts of protein were denatured with loading buffer (Beyotime) at 100°C for 10 min, then loaded onto a 12% SDS-PAGE gel for electrophoresis and transferred to a methanol-activated polyvinylidene fluoride membrane (Millipore, USA). The membrane was blocked in tris-buffered saline (TBS) containing 5% bovine serum albumin (MP Biomedical, USA) for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. Bod1 (1:1000, ABGENT, China) and GAPDH (1:1000, ZSGB-BIO, Beijing, China) primary antibodies were used. After washing with TBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature. Secondary antibodies conjugated with HRP included Goat-Anti Rabbit IgG (1:5000, ZSGB-BIO) and Rabbit-Anti Goat IgG (1:5000, ZSGB-BIO). Relative protein levels were quantified using Image J software.

2D- and 3D-grown cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The total protein concentrations of the lysates were determined using a protein assay kit (Bio-Rad, USA). Equal amounts of protein were denatured with loading buffer (Beyotime, Shanghai, China) at 100°C for 10 min, then loaded in 12% SDS-PAGE for electrophoresis, and transferred to a methanol-activated polyvinylidene fluoride membrane (Millipore, USA). The membrane was blocked in tris-buffered saline (TBS) containing 5% bovine serum albumin (MP Biomedical, USA) for 2 h at room temperature. Primary antibodies were incubated overnight at 4°C. The primary antibodies included: OCT4 (1:1000, Abcam, USA), SOX2 (1:1000, CST, USA), NANOG (1:1000, ABGENT, USA), β-catenin (1:1000, CST, USA) and GAPDH (1:1000, ZSGB-BIO, Beijing, China). After washing with TBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)- labeled secondary antibody for 1 h at room temperature. Secondary antibodies conjugated with HRP include: Goat-Anti Rabbit IgG (1:5000, ZSGB-BIO, Beijing, China) and Rabbit-Anti Goat IgG (1:5000, ZSGB-BIO, Beijing, China).