Zf 0317

According to the manufacturer’s procedures, we performed immunohistochemistry in paraffin-embedded eyeballs and immunofluorescence staining in ARPE-19 cells. Next, SirT1 mouse mAb (1F3, Cst, USA, 1:100) and anti-p53 antibody (PAb240, Abcam, United Kingdom, 1:500) were incubated at 4°C overnight. Antibody dilution buffer (1× PBS/1% BSA/0.3% Triton™ X-100) was used to dilute the fluorescent substance-labeled secondary antibody (ZF-0317, Zsgb-Bio, China, 1:400). Then, retinal tissue and cells were incubated for 1–2 h and protected from light. Immunofluorescence images were acquired using an inverted fluorescence microscope (ECLIPSE Ti2, Nikon, Japan).

Retinal tissue and ARPE-19 cells in each group were lysed using RIPA lysis buffer (P0013B, Beyotime, China), supplemented with protease inhibitor (410090, Bimake, United States) and phosphatase inhibitor (510042, Bimake, United States) on ice. The liquid supernatant was analyzed using the BCA method (P0010, Beyotime, China), followed by electrophoresis, membrane transfer (PVDF, Bio-Rad), and antibody incubation ((SirT1 mouse mAb, 1F3, Cst, USA), (anti-p53 antibody, PAb240, Abcam, United Kingdom), (acetyl-p53 antibody, Lys382, Cst, United States), (antibodies against acetyl-Lysine, #9441, Cst, United States), and (GAPDHXP® Rabbit mAb) (D16H11, Cst, United States)) overnight at 4°C. After that, secondary antibodies ((anti-rabbit IgG, HRP-linked antibody, #7074, Cst, USA) and (rhodamine-labeled rabbit anti-goat IgG, ZF-0317, Zsgb-Bio, China)) were added and gently shaken at room temperature for 2 h. Protein samples were visualized using an ECL chemiluminescence kit (A + B, Bio-Rad, USA) (Supplementary Material S3).