Protein Extraction and Analysis from Mouse Intestinal Crypts

Based on the previous study [21 (link)], 200 μL Radioimmunoprecipitation (RIPA) solution (Beyotime, Shanghai, China), which contains 1x protease inhibitor, was added to crack the crypts and shaken at 4 °C for 20 min to fully crack the crypts. The protein concentration was determined by Bicinchoninic Acid (BCA) kit (Beyotime, Shanghai, China). After adding the protein loading buffer, the protein was denatured at 100 °C for 10 min and stored at −80 °C refrigerator. The protein samples with the calculated concentration were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel electrophoresis. Then, they were gummed, transferred, sealed, and incubated primary antibody overnight at 4 °C. Protein primary antibody information and dilution ratio: BAX (1:1000, Proteintech, Wuhan, China), Caspase9 (1:1000, CST, Boston, MA, USA), PCNA (1:1000, Abcam, Cambridge, UK), Olfm4 (1:1000, CST, Boston, MA, USA), P-YAP (1:1000, Abclonal, Wuhan, China), YAP (1:1000, Abcam, Cambridge, UK), Cyclin D1 (1:1000, CST, Boston, MA, USA). Finally, we incubated secondary antibodies, and the ECL solution was used to reveal the bands. A total of 30 experimental animals C57BL/6 mice were used.