E bc r347

For western blotting analysis, the rabbit anti-hOGG1 and hAAG polyclonal antibodies (ZIKER-3687R and 2412R, ZIKER Bio, Shenzhen, China) were used against hOGG1 and hAAG expressed in A549 and HeLa cells, respectively. After cancer cells (5 × 10⁶) were collected, hOGG1 and hAAG enzymes were extracted from the nucleus and cytoplasm in A549 and HeLa cells, respectively, using the nuclear extract kit, and the resultant supernatants were analyzed by western blotting. With histone H3 (RLM3038, RuiYing Bio, Wuhan, China) and actin (GB12001, Servicebio, Wuhan, China) as the internal reference proteins, the levels of hOGG1 and hAAG enzymes from different parts of A549 and HeLa cells were evaluated using the western blot detection kit (E-IR-R304A, Elabscience, Wuhan, China). The immune complexes were detected by an excellent chemiluminescent substrate detection kit (E-BC-R347) (Elabscience, Wuhan, China), and the intensities of protein strips were measured by an Epson V300 scanner (Epson, Suwa, Japan) and quantified by Alpha Ease FC software (Alpha Innotech, San. Leandro, CA, USA).

Western blot was performed as described previously [17 (link)]. RIPA buffer (R0278, Sigma–Aldrich, USA) was applied to lyse and extract the total protein from cells. After the protein concentration was measured by BCA protein quantitation kit (55R-1544, Fitzgerald, USA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were exploited to separate the protein (45 μg) and marker (5 μL; G2086, Servicebio, China), which was then moved to polyvinylidene fluoride membranes (24937, Sigma-Aldrich, China). The membranes were sealed by defatted milk and subsequently incubated with primary antibodies (Table 2) at 4°C overnight. Next, the membranes were incubated with the goat anti-rabbit (ab97051, 1 : 5000, Abcam) or rabbit-anti-mouse secondary antibody (ab6709, 1 : 2000, Abcam) for 2 h. An excellent chemiluminescent substrate detection kit (E-BC-R347, Elabscience, China) was used to measure the protein bands, and an eZwest Lite auto western blotting system (Genscript, Piscataway, NJ, USA) was employed to scan the bands.