Proteins in ovaries were extracted using RIPA buffer (Beyotime Biotechnology, Haimen, China) and protease inhibitors (Sigma Aldrich, St. Louis, MO, USA), 30 μg protein was electrophoresed.
The gel was electrotransferred to a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany), blocked with 5% nonfat milk for 90 min, and the membrane was then incubated with
primary antibodies (CHOP, 1:500 dilution, 60303-1-1g, Proteintech; β-actin, 1:5000 dilution, SC-47778; Santa Cruz Biotechnology) overnight at 4ºC. Subsequently, secondary antibody conjugated
with horseradish peroxidase (1:5000; ZB-2305; ZSGB-BIO) was used to detect the primary antibody that had bound to the membrane. The signals were obtained and analyzed using the
chemiluminescence kit (Millipore, MA, USA) and Quantiscan software (Biosoft, Cambridge, UK) [26 (link), 36 (link), 37 (link)].
The gel was electrotransferred to a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany), blocked with 5% nonfat milk for 90 min, and the membrane was then incubated with
primary antibodies (CHOP, 1:500 dilution, 60303-1-1g, Proteintech; β-actin, 1:5000 dilution, SC-47778; Santa Cruz Biotechnology) overnight at 4ºC. Subsequently, secondary antibody conjugated
with horseradish peroxidase (1:5000; ZB-2305; ZSGB-BIO) was used to detect the primary antibody that had bound to the membrane. The signals were obtained and analyzed using the
chemiluminescence kit (Millipore, MA, USA) and Quantiscan software (Biosoft, Cambridge, UK) [26 (link), 36 (link), 37 (link)].
