Enhanced ecl reagent

Cells of different treatment were collected by centrifugation with 1200 rpm in 5 min, processed and lysed in RIPA buffer supplemented with a protease inhibitor cocktail and PMSF (all purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer instructions. Protein concentration was determined by BCA protein assay (CoWin Biotech Co., Ltd, Beijing, China), and 5 µl of protein were loaded and separated by 8 to 12% SDS-PAGE. After transfer to PVDF membranes, 5% BSA in TBST was used to block the membrane at room temperature for 1 h. The membranes were incubated with primary antibodies [ACSS2 (at 1:2,000 dilution); ATM, p-ATM, BRCA1, p-BRCA1, Bcl-xL, γH2AX, DNA-PKcs, ULK1, p-ULK1, AMPK, p-AMPK, PCNA, and Ki-67 (at 1:1,000 dilution); β-actin (at 1:2,000 dilution); Bax (at 1:500 dilution)] saturated with 5% BSA in TBST at 4°C overnight. On the next day, the membranes were incubated with HRP-conjugated anti-rabbit or -mouse antibody (at 1:5,000 dilution, product #7074 and #7076; Cell Signaling Technology) for 1 h at room temperature, and then exposed to enhanced ECL reagent (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China), imaged by an automatic ChemisScope-4300 imager (Clinx Science Instruments Co., Ltd., Shanghai, China) and data were analyzed with Fluor Chem FC3 software (Protein-Simple, USA).