Beyoecl plus developer

D283 and D341 cells were lysed with protease inhibitor, and RIPA cleavage buffer (Thermo Fisher Scientific, Shanghai, China). The protein lysates were resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and imprinted on polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) for analysis. Anti-CENEP (1:1,000 dilution, DF7745; Affinity Biosciences, OH, USA), anti-P53 (1:500 dilution, AF0879; Affinity Biosciences, OH, USA), anti-P21 (1:500 dilution, AF6290; Affinity Biosciences, OH, USA), anti-CDK1 (1:500 dilution, DF6024; Affinity Biosciences, OH, USA) and anti-β-Actin (1:5,000 dilution, AF7018; Affinity Biosciences, OH, USA) were incubated overnight. Goat Anti-Rabbit IgG (H+L) HRP-conjugated secondary antibodies (1:5,000 dilution; S0001, Affinity Biosciences, OH, USA) were added at room temperature for 2 h. The membrane was detected with BeyoECL Plus developer (Beyotime, Shanghai, China).

After properly processing the cells, the cells were placed on ice before adding RIPA buffer (50 mM Tris, pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride (EDTA, leupeptin) with protease inhibitor (100 mM) at a proportion of 99:1. The protein lysates were quantified by BCA method, separated by 5%-15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Abcam, Cambridge, MA, USA). Subsequently, the membrane was sealed with 5% non-fat milk in Tris buffer saline for 90 min at room temperature. The membrane was then washed and incubated overnight at 4 ℃ with an appropriate primary antibody at 1:1000. The next day, the membrane was washed and incubated with horseradish peroxidase-coupling secondary antibody in Tris buffer saline at room temperature for 1 h. At last, the membrane was developed with BeyoECL Plus developer (Beyotime, Shanghai, China) using the Bio-Rad Molecular Imager FX.

The cells are properly treated and placed on ice before adding RIPA buffer containing protease inhibitor cocktail (ratio, 100:1) (Roche Diagnostics Corp. Indianapolis, IN, United States). The protein concentration was examined by the BCA method (ab102536; Abcam), separated by 8%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to a polyvinylidene difluoride membrane (Abcam, Cambridge, MA, United States). Subsequently, the membrane was blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20 (TBST) for 90 min at room temperature. Then the membrane was washed with TBST and incubated at 4°C overnight with the primary antibody in a ratio of 1:1,000. The next day, the membrane was washed with TBST and followed by incubation with horseradish peroxidase-coupling secondary antibody (goat anti-rabbit) at room temperature for 2 h. At last, the membrane was washed with TBST and detected with BeyoECL Plus developer (Beyotime, Shanghai, China) using the Bio-Rad Molecular Imager FX.