Enhanced bicinchoninic acid assay kit

Total protein was extracted using RIPA lysis buffer (Thermo Scientific, United Kingdom), and its concentration was determined with an enhanced bicinchoninic acid assay kit (CWBio, China). Around 40 mg protein per sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (CWBio, China). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight with primary antibodies targeting MRTO4 (1:2000, ab212044, Abcam, United States), BOP1 (1:3000, ab32053, Abcam, United States), PES1 (1:5000, ab56701, Abcam, United States), NDUFS8 (1:3000, ab226760, Abcam, United States), NDUFS6 (1:3000, ab226760, Abcam, United States), NDUFA8 (1:3000, ab226760, Abcam, United States) and β-actin (1:5000, ab8226, Abcam, United States) at 4 °C. The membranes were washed thrice with TBST (Tris-buffered saline with Tween 60), and probed with horseradish peroxidase-conjugated secondary antibody (1:5000) for 1.5 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence system, and the membranes were exposed to X-ray films (Bio-Rad, United States). Densitometric analysis was performed using Image Pro-Plus software (Media Cybernetics, United States), and relative protein expression levels were normalized to β-actin.

Total protein was extracted from GC and paracancerous tissues using RIPA lysis buffer (Pierce, Thermo Scientific, Cramlington, United Kingdom)[13 ]. Protein concentration was determined with an enhanced bicinchoninic acid assay kit (CWBio, Beijing, China). A total of 40 mg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (CWBio, Beijing, China). After blocking with 5% non-fat milk for 1 h at room temperature (RT), the membranes were incubated overnight at 4 °C with primary antibodies directed to WISP1 (Abcam, ab178547), XRCC1 (Abcam, ab9147), γH2AX (Abcam, ab2893), and β-actin (Abcam, ab8226). After washing three times with TBST (Tris-buffered saline with Tween 60), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at 1:5000 dilution for 1.5 h at RT. Protein bands were visualized using an enhanced chemiluminescence system, and exposure of the membranes to X-ray films (Bio-Rad, Hercules, CA, United States). Densitometric analysis was performed using Image Pro-Plus software (Media Cybernetics, United States). Relative protein expression levels were normalized to β-actin.