Ripa buffer

The cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Selleckchem). The protein concentration was tested with a BCA kit, and appropriate amounts of protein were prepared for SDS-PAGE and then transferred to PVDF membrane (Millipore, MA, USA). The membranes were blocked for 1 h with 5% non-fat dry milk and then incubated with rabbit anti-SIRT1 (1:1000; #9475; Cell Signaling Technology Europe, Netherlands); TFAM (1:1000; #8076; Cell Signaling Technology Europe, Netherlands); CATALASE (1:1000; #12,980; Cell Signaling Technology Europe, Netherlands); SOD2 (1:1000; #MA1-106; Invitrogen); UQCRC2 (1:1000; #ab14745; Abcam); MTCO2 (1:1000; #ab110258; Abcam); ASCL4 (1:500; #sc-365230; Santacruz); Actin-b (1:5000, A5441; Sigma) mABs were used as loading controls. The results were imaged using a gel image analysis system (Bio-Rad, California, USA) according to the manufacturer’s instructions.

Total cell lysates were obtained using RIPA buffer (CWBIO) containing protease inhibitor PMSF (Selleck) and dithiothreitol (DTT) (Sigma). Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The bands were visualized using the ECL chemiluminescence kit (Monad). Nuclear-cytoplasmic fractions were obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (catalog no. P0027; Beyotime), then subjected to western blotting. Antibodies used are shown in Supplementary Table S2.

The cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Selleckchem). For histone extraction, cells were lysed with NETN buffer containing protease and phosphatase inhibitors, and histones were extracted with HCL. The protein concentration was tested with a BCA kit, and appropriate amounts of protein were prepared for SDS-PAGE and then transferred to PVDF membrane (Millipore, MA, USA). The membranes were blocked for 1 h with 5% nonfat dry milk and then incubated with rabbit anti-p-ATM mAb (Ser1981; 1:1000; #5883; CST) and rabbit anti-p-ATR mAb (Ser428; 1:1000; #2853; CST), mouse anti-P53 (1:500; sc-47698; Santa Cruz) mAb, rabbit anti-ATR mAb (1:1000; #13934; CST), rabbit anti-ATM mAb (1:1000; #2873; CST), rabbit anti-PARP mAb (1:1000; #9532; CST), rabbit anti-Bcl2 mAb (1:1000; Cat. No. 12789-1-AP; Proteintech), rabbit anti-Bax mAb (1:1000; Cat. No. 50599-2-lg; Proteintech), rabbit anti-r-H2AX mAb (Ser139; 1:1000; #9718; CST), rabbit anti-P21 mAb (1:1000; Cat. No. 10355-1-AP; Proteintech), rabbit anti-H2AX mAb (1:500; D155226-0025; Sangon Biotech). Additionally, α-tubulin (1:1000; #5335; CST) and GAPDH (1:3000; Cat. No. 60004-1-lg; Proteintech) were used as loading controls. The results were imaged using a gel image analysis system (Bio-Rad, California, USA) according to the manufacturer’s instructions.