Claspin

TMAs were purchased from Shanghai Biochip Co., Ltd., (Shanghai, China). The histopathological diagnosis was determined by the World Health Organization criteria. Tumor staging was defined according to the 6th edition of the tumor-node-metastasis (TNM) classification of the International Union Against Cancer. Overall survival (OS) was termed as the interval between the dates of surgery and death or final follow-up.
Immunohistochemistry staining of paraffin sections were manufactured on 4 µm-thick slides, followed by EnVision two-step procedure of Dako REAL™ EnVision™ detection system (Dako, Carpinteria, CA, USA). After antigen retrieval, the slice was incubated with the primary antibodies USP20 (1:250; Proteintech Group, Chicago, IL, USA) and Claspin (1:1500; Proteintech Group) overnight at 4°C, followed by incubation with the HRP labeled secondary antibodies at 37°C for 0.5 h and slide was visualized by diaminobenzidine.

MKN45, NCI-N87, BGC-823 and MGC-803 cells (5×10⁵/well) were cultured overnight in 6-well plates and transfected as described above. Two days later, the cells were harvested using mammalian protein extraction reagent (Pierce, Rockford, IL, USA) mixed with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The cell lysates (50 µg/lane) were separated on 10% SDS-PAGE and transferred to a polyvinylidene di fluoride membrane. After blocking with 5% non-fat milk in PBST buffer for 2 h, the membranes were incubated overnight at 4°C with the primary antibodies for USP20 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Claspin (1:1,000; Proteintech Group), ATG5 (1:1,000; Cell Signaling Technology, Boston, MA, USA), LC3 (1:1,000; Cell Signaling Technology) or GAPDH (1:2,500; Santa Cruz Biotechnology) and GAPDH was used as loading control. Secondary antibodies (1:10,000; LI-COR Biosciences, Lincoln, NE, USA) and infrared imaging system (LI-COR Biosciences) were applied to visualize the protein bands.