Ab189494

The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated with Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4 (1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), Rabbit anti-NF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse anti-GAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C, and secondary anti-mouse HRP-conjugated (1:10,000, Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-rad, 170-6515) were incubated for 2 h at room temperature. The signals were visualized using ECL chemiluminescence reagent (Beyotime; US Everbright Inc.).

Protein was extracted with Western Cell Lysis Buffer with 1% phenylmethyl sulfonyl fluoride (Sangon Biotech, Shanghai, China). Next, a modified BCA Protein Assay Kit (Sangon Biotech, Shanghai, China) was used to determine the protein concentration according to the absorbance at 562 nm. The 30 μg total protein was loaded onto an SDS polyacrylamide gel (Solarbio, Beijing, China), including 5% stacking gel and 8% resolving gel. After electrophoresis at 75 V for 30 min and 100 V for 60 min, protein was transferred to a 0.45 μm PVDF membrane (Millipore, Billerica, MA, United States) at 100 V for 2–4 h, blocked in 5% non-fat milk for 60 min, and incubated with primary antibodies for 12 h at 4°C (Abcam, UK) (FOXO1 69KD, 1:1,000, ab179450; sirtuin 1 (SIRT1) 110KD, 1:1,000, ab189494; adipose triglyceride lipase (ATGL) 55KD, 1:1,000, ab109251; sterol regulatory element-binding protein-1c (SREBP-1c) 127KD, 5 μg/ml, ab3259; fatty acid synthase (FASN) 273KD, 1:1,000, ab128856; GAPDH 37 KD, 1:10,000, ab181602); CYP2E1 (57KD, 1:2,000, 19937-1-AP) (Proteintech, UK), and the HRP conjugated secondary antibody (Beyotime, Shanghai, China) for 60 min. Subsequently, a BeyoECL Star kit (Beyotime, Shanghai, China) was used to conduct the western blotting detection, and the relative quantitative analyses were performed in Image J software.

For immunohistochemical experiments, paraffin sections were first dewaxed and dehydrated in xylene using 0.1% trypsin to repair the antigen at 37 °C for 15 min and was then incubated in 3% hydrogen peroxide solution for 15 min to inhibit peroxidase activity. The sections were blocked with Goat serum for 30 min and then incubated with the primary antibody overnight at 4 °C. Primary antibodies include: Anti-SIRT1: (Abcam, 1:200, ab189494), Anti-β-catenin:(Proteintech,1:200,51,067–1-AP), Anti-LEF1:(proteintech,1:200,14,972–1-AP), Anti-MMP13:(abcam,1:300,ab39012), and Anti-collagen II: (abcam,1:300,ab34712). The number of positive cells was counted with Image pro-plus 6.0 software. The sections were fixed in 4% PFA for immunofluorescence experiments and then combined with the corresponding antibody at 4 °C overnight, followed by a combination with Alexa Fluor 488 goat anti-rabbit secondary antibody (Abcam, 1:300, ab150077) and incubated at 37 °C for 60 min.
An inverted fluorescence microscope was used to observe the positive cells, and Imagepro-plus 6.0 software was used to count the proportion of positive cells.