Pp2a cα β

Cells were harvested by scraping or trypsinization and lysed with buffer [50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1X protease/phosphatase inhibitor cocktail (Thermo 1861280)]. 2X SDS loading buffer was added to the lysates before heating at 95°C for 5 min.
Three to eight percentage Tris-Acetate SDS PAGE gradient gels (Invitrogen EA0378) were used to resolve ATR-H from ATR-L; otherwise, standard SDS PAGE gels were used. PVDF membranes (Amersham 10061-492) were used to capture proteins transferred from gels. Chemiluminescent signal was captured using the GE Amersham Imager 680. Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

The lysate buffer of the protease inhibitor mixture and the 1% phosphatase inhibitor mixture were added to gastric cancer cells and lysed on ice for 10 minutes to extract the proteins. Protein concentration of each group was quantitatively detected by protein quantitative kit (BCA method, Beyotime Biotechnology, Shanghai, China). Before the WB experiment, protein lysates were added into the 5× SDS-PAGE protein loading buffer in proportion, boiled for 10 minutes, and stored in the refrigerator at -20°C. Protein extract (30-50 μg) was extracted for precast gel electrophoresis. After electrophoresis, the PVDF membrane (Beyotime Biotechnology, Shanghai, China) was transferred and then sealed with 5% skimmed milk powder at room temperature for 1 hour. After slight rinsing of the blocking solution with TBST, the diluted primary antibody was incubated overnight at 4°C, and the next day with the corresponding diluted secondary antibody was incubated at room temperature for 1 h. Finally, an appropriate amount of developer solution was added in a dark room before exposure strips were performed. The primary antibodies used in this study were PP2A-Cα/β (1 : 500; Santa Cruz Biotechnology, Dallas, TX, USA); METTL3 (1 : 1000; Proteintech Group, Chicago, IL, USA); GAPDH (1 : 1000; Proteintech Group, Chicago, IL, USA); and β-actin (1 : 1000; Proteintech Group, Chicago, IL, USA).