P chek1

The Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor cocktail (Beyotime, Shanghai, China) was used to extract cellular protein. The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) was use to extract subcellular protein. The extracted samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis in order to separate the component proteins before they were immunoblotted onto PVDF membranes. 5% nonfat dry milk in TBST was used to block membranes. The appropriate primary antibodies were then added to the membranes and allowed to incubate. This was followed by incubation with HRP-conjugated secondary antibodies. Western blotting analysis was performed using anti-RACGAP1, LIG3, γH2A.X, p-ATM, p-ATR, p-CHEK1 and p-CHEK2, PARP1, caspase3 and c-caspase3, PAR, GAPDH and Histone H3 antibodies (p-ATM, p-ATR, p-CHEK1, p-CHEK2 and PARP1 were supplied by ABclonal, MA, USA, others were from Abcam, MA, USA).