Proliferating cell nuclear antigen (pcna)

Cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) and phosphate protease inhibitor. Protein samples were harvested, boiled, separated with 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2α (1 : 1000, ABclonal, Wuhan, China), eIF2α phosphorylation (p-eIF2α, 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China). Then, they were incubated in horseradish peroxidase-conjugated secondary antibodies (1 : 10000, Biosharp, Hefei, China) and visualized with an electrochemiluminescence reagent.

The whole proteins from frozen ilea or cells were extracted on ice by using RIPA buffer (Beyotime Biotechnology) with addition of phosphatase inhibitor cocktail and protease inhibitor cocktails (Bimake). Proteins of 50 μg from tissues or 30 μg from cells were resolved by 10% SDS‐PAGE for further experiments, as described before.⁷ (link) Primary antibodies included CDK4 (Proteintec), P21 (SAB, College Park), COX‐2 (Abcam), c‐MYC (Abcam), cyclin D1 (Huabio), PCNA (Huabio), cleaved‐caspase 3 (CST, Danvers), active β‐catenin (CST), total β‐catenin (CST), total GSK3β (CST), phospho‐GSK3β (CST) and GAPDH (ABclonal).