Bca protein quantitation kit

A protein extraction kit (KeyGen Biotech Co. Ltd., Nanjing, China) was used to extract protein from tumor tissue, and the protein concentration was determined using a BCA protein quantitation kit (KeyGen Biotech Co. Ltd., Nanjing, China). Thirty micrograms of protein were separated by 10% SDS PAGE, then transferred to PVDF membranes (Millipore, USA). After blocking with 5 percent skim milk, the membranes were incubated with antibodies against GAPDH (1:2,000 cat. no.TA-08; BIOSS, Beijing, China), BAX (1:1,000 cat. no.60267-1-Ig proteintech), and Bcl-2 (1:1,500 cat. no.26593-1-AP proteintech) antibodies at 4°C overnight. Then, the membranes were incubated with goat anti‑mouse or rabbit IgG horseradish peroxidase (HRP)‑conjugated secondary antibodies (1:5,000; cat. nos. A21010 and A21020; Abbkine Scientific Co., Ltd.) for 1 h at room temperature, and signals were detected by ECL chemiluminescence and measured using ImageJ software.

Freshly isolated GCs from 4-week-old mice (n = 8 for each genotype) were seeded in 12-well microplates at a density of 5 × 10⁵ cells/well and cultured until confluent and cultured in McCoy’s 5a medium with 10% FBS. The culture medium was replaced by serum-free medium supplemented with 10 μg/mL insulin, 5.5 μg /mL transferrin, and 5 ng/mL selenium when cell density reached 80%. After 12-h culture, serum-free medium supplemented with or without 100 mIU/mL FSH and/or 1 μM testosterone (Sigma-Aldrich) replaced the previous medium. Forty-eight h later, the medium was collected and centrifuged. The estradiol and progesterone concentrations in the supernatant were measured using mouse estrogen and progesterone ELISA kits (Cusabio Biotech Co., Wuhan, China), respectively, according to the instructions (Sun et al., 2014 (link)). Meanwhile, cells in each well were divided into two equal portions. One portion was used to extract total RNA, and the other was used to measure the total protein content with a BCA protein quantitation kit (KeyGEN, Nanjing, China). The estradiol and progesterone levels were normalized by the ratios of estradiol/progesterone concentration to total protein content. Four samples were measured for each group, and the assay was repeated twice.

Antibodies against LC3, Beclin-1, p62, Cleaved-PARP, Cytochrome c, Bax, Bcl-2, ERK/pERK1/2, caspase 9, p-p70S6K, mTOR/p-mTOR, E-cadherin, p-4EBP1, caspase 3, EpCAM, N-cadherin, GAPDH, COX IV and β-Tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). BCA Protein Quantitation Kit and Annexin V-FITC/PI Apoptosis Detection Kit were obtained from KeyGen Biotech (Nanjing, China). Other reagent purchase information were as follows: Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA), Cell Counting Kit-8 (Meilun Biotechnology, Dalian, China), ROS Assay Kit (Beyotime Biotechnology, Haimen, China), Acridine Orange (Absin Bioscience, Shanghai, China).