Acetyl foxo1

The protein was extracted from hippocampus tissues using RIPA lysis buffer (1:100; Beyotime, Shanghai, China). Equal amounts of protein were separated using the SDS–polyacrylamide gel (SDS–PAGE) gel preparation kit (Solarbio). Electrophoresis was carried out on SDS–PAGE using 20 μg of total protein in each lane. After electrophoresis, the protein was transferred to a PVDF membrane. Then, the membrane was incubated with corresponding primary antibodies (Sirt1, 1:2000; Bioworld; PI3K, 1:1000; Bioss; AKT, 1:2000; Cell Signaling; p‐AKT, 1:1000; Cell Signaling; Gabarapl1, 1:1000; Bioss; FoxO1, 1:1000; Bioss; ATG12, 1:1000; Abcam; acetyl‐FoxO1, 1:2000; Thermo Fisher; p‐FoxO1, 1:1000; Bioss; GAPDH, 1:2000; Zsbio; LaminB1, 1:1000; Bioss) at 4°C overnight and the secondary antibody (1:20,000; Abcam) for 1 h, respectively. Eventually, the targeted antigens were detected by standard chemical luminescence methods. Band intensities were measured with Image software. During the experiments, we first detected the protein expression levels of Sirt1, PI3K, AKT, p‐AKT, FoxO1, ATG12, acetyl‐FoxO1, p‐FoxO1, and Gabarapl1 and then detected the protein expression levels of p‐FoxO1 in the cytoplasm and FoxO1 in the nucleus.

Western blot analysis was carried out as described previously [9 (link), 17 (link)]. Briefly, the lung tissue protein or cellular protein was separated by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred onto polyvinyl difluoride membranes. The membrane was blocked with 5% fat-free dry milk in tris-buffer solution Tween, the blots were incubated with primary antibodies SIRT1 (Cell Signaling Technology, 9475), Parkin (Cell Signaling Technology, 2132), LC3-II (Cell Signaling Technology, 3868), PINK1 (Abcam, ab186303), Acetyl-FoxO1 (Thermo Fisher Scientific, PA5104560) at 4 °C overnight. Then, the membranes exposed to the corresponding horseradish peroxidase-conjugated secondary antibody for 2 h. The membranes were visualized with diaminobenzidine staining and exposed to film. VDAC (Cell Signaling Technology, 4661) and GADPH (Cell Signaling Technology, 5174) were used as the internal control for loading variations. Band intensities were analyzed with Image J software.