Anti p53

Cells were lysed with radio immunoprecipitation assay buffer (RIPA) containing 1 mmol/L PMSF (Solarbio, China). Protein extractions were separated by 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were incubated overnight at 4 °C with primary antibodies specific for anti-β-actin, anti-CDK2, anti-CyclinD1, anti-CyclinE, anti-PCNA, and anti-P53 (1:1,000; Wanleibio), anti-Bcl-2, anti-Bax, anti-Caspase9, anti-FOXC1, and anti-MyoD1 (1:1,000; Abways Technology), anti-MyoG, (1:1,000; Abcam, Cambridge, England). After incubation with secondary antibodies, signals were detected using a chemiluminescence system (Bio-Rad, USA).

Granulosa cells were incubated with 3-NPA at 5.0 mmol/l for 24 h and then were harvested. The total protein was extracted using RIPA Lysis Buffer and 10% phenylmethanesulfonyl fluoride (Beyotime, China). Protein concentration was determined by using a BCA Protein Assay Kit (Beyotime, China). Approximately, the same amount of protein was separated using 10–12% SDS-PAGE, transferred electrophoretically onto the polyvinylidene membrane (Bio-Rad) and blocked with 5% nonfat dry milk. The primary antibodies used in the present study were anti-Caspase 3 (CST, U.S.A.), anti-Bcl-2 (Wanlei Biotechnology, China), anti-p53 (Wanlei Biotechnology, China), anti-Bax (Bioss Biotechnology, China), anti-β-actin (TransGen Biotechnology, China). The membrane was incubated with the primary antibody solution overnight at 4°C, and then washed four times with the TBST (TBS, 0.1% Tween 20). The corresponding secondary antibody (1:5000) was added and incubated at room temperature for 1 h. The protein bands were visualized by using the BeyoECL Plus (a chemiluminescence reaction; Beyotime, China) in an Image Lab software (Bio-Rad, U.S.A.). The bands were quantified using an ImageJ software (NIH, U.S.A.).

We collected the treated HEK293 cells, washed them once with PBS, added the proteolytic lysate, and lysed the cells on ice. The cells were centrifuged at 4 ℃ and 13,000 r/min for 10 min. Store in tube at − 80 °C. The BCA protein concentration test was used to calculate the protein concentration of the sample. Separated on a 10% SDS polyacrylamide gel. It is then electrically transmitted to the PVDF membrane. Before incubating the primary antibody, tailor the PVDF membrane according to the molecular weight range of the target antibody. Use anti-HO-1 (wanleibio, China), anti-Nrf2 polyclonal (wanleibio, China), anti-NF-κB, anti-NQO1 (Solarbio, China), anti-keap1 (wanleibio, China), anti-P53 (wanleibio, China), anti-Lamin B, anti-caspase-3 (Solarbio, China) and cleaved caspase-3 (Solarbio, China) for western blotting. Finally, the cut PVDF membrane was placed on a plastic wrap and an appropriate amount of ECL kit was taken. Medium volumes of liquids A and B were mixed, mixed and added to the surface of the membrane, transferred to a gel imaging analyzer, and exposed in a chemical light-sensitive mode. After exporting photos in TIF format, analyze the optical density of each band under ImageJ software^{18 (link)}.