Bicinchoninic acid kit

The total protein was extracted from the cells and the protein concentration was determined by bicinchoninic acid kit (AmyJet Scientific, Wuhan, Hubei, China). The extracted protein was mixed with the loading buffer and centrifuged after boiling at 95 ℃ for 10 min, separated with 10% polyacrylamide gel electrophoresis, and transferred to membrane. The membrane was sealed with 5% skim milk in tris-buffered saline with tween 20 (TBST) for 1 h, added with primary antibody against SOX2 (1: 1000, Abcam, Cambridge, MA, USA), OCT4 (1: 1000), Nanog (1: 1000) (Cell Signaling Technology, Beverly, MA, USA), CD63 (1: 200), CD81 (1: 200) and GAPDH (1: 1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 ℃. The protein was added with the corresponding secondary antibody (1: 2000, Abcam, Cambridge, MA, USA) for 1 h at 37 ℃ and developed by chemiluminescence reagent. The protein imprinted image was analyzed with ImageJ2x Software (National institutes of health, Maryland, USA).

Tissue samples (15–20 mg) were placed in the centrifuge tube, ground 50–60 times with a plastic stick, added with 200 μL of natural cell lysate and ground 30–60 times. The samples were incubated on ice for 5 min and centrifuged at 14,000–16,000 rpm at 4 °C for 1–2 min. Then, the tube was placed on ice, and protein was collected. Protein concentration was determined by a bicinchoninic acid kit (AmyJet Scientific, Wuhan, Hubei, China). The extracted protein was separated by 10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked by 5% skim milk and incubated with primary antibodies IGF1 (1:1000), caspase-3 (1:500), Cleaved caspase-3, Bax, Bcl-2, and GAPDH (1:1000, Cell Signaling Technology, Beverly, MA, USA). After that, the membrane and the secondary antibody (1:5000; Thermo Fisher Scientific) were incubated for 1 h and the protein bands were visualized by the enhanced chemiluminescence kits (Pierce, Rockford, IL, USA). GAPDH was the internal control and protein band images were analyzed by ImageJ2x V2.1.4.7 (Rawak Software, Inc., Germany). This method is also suitable for cell experiments.