Western Blotting Protocol for Protein Analysis

Western blotting was performed as described previously [34 (link)]. Briefly, 20 μg samples were loaded and separated on 10% or 12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% milk in TBST for 1 h, we used TBST to dilute primary and secondary antibodies. Membranes were incubated with primary antibody overnight at 4°C, washed in TBST, and incubated with HRP-conjugated secondary antibody for 60 min. The proteins of interest were performed using an ECL western blot detection kit (Bio-Rad). ImageJ software was used to evaluate the densitometry. Actin or proliferating cell nuclear antigen (PCNA) was used as loading control. Antibodies used were as follows: p53 (Beyotime, #AF7671), PAI-1 (Cell Signaling, #49536), p21 (Cell Signaling, #2947), SIRT1 (Cell Signaling, #8469), SIRT5 (Cell signaling, #8779), NRF2 (ABclonal, #A0674), PCNA (Cell Signaling, #13110), and Actin (Cell Signaling, #3700).