Protease and phosphatase inhibitors

The liver tissues were homogenized and lysed in ice-cold lysis buffer supplemented with protease and phosphatase inhibitors (Topscience, Shanghai, China) and centrifuged at 12,000 g for 20 min at 4°C, and then the clarified supernatants were collected. Then proteins were quantified with the Pierce BCA Protein Assay Kit (Thermo, United States) assay according to the manufacturer’s instructions. The western blot was performed as previously described (Li et al., 2022a (link); Li et al., 2022b (link)). Briefly, after SDS-PAGE and transmembrane, the target proteins were accordingly probed with first antibodies against GAPDH (10494-1-AP, 1:1,000, Proteintech), phospho-Stat3 (9145, 1:1,000, CST), Stat3 (9139, 1:1,000, CST), phospho-p38 MAPK (4511, 1:1,000, CST), p38 MAPK (8690, 1:1,000, CST), phospho-NF-κB p65 (3033, 1:1,000, CST), NF-κB p65 (6956, 1:1,000, CST), phospho-IκBα (2859, 1:1,000, CST), and IκBα (4814, 1:1,000, CST), respectively. After incubation with the corresponding HRP-conjugated secondary antibody, the signal of the target protein was detected using a ChemiDo XRS gel imager system (Bio-Rad, United States) with Immobilon Western Chemiluminescent HRP Substrate (Millipore, United States) and was scanned by ImageJ software. The ratio of the target protein was normalized to the internal control protein GAPDH, and fold-change was calculated relative to the control group.

Western blot analysis was conducted using routine protocols. Briefly, hippocampal and cortical tissues were independently homogenized with RIPA buffer (New Cell and Molecular Biotech Co., Ltd., Suzhou, China) containing protease and phosphatase inhibitors (Topscience, Shanghai, China), after which they were centrifuged at freezing temperatures at 13000 g for 15 minutes. Total protein concentration was measured using a BCA assay (CWBio, Beijing, China). Twenty μg protein was analyzed in each assay, as described in the published literature [16 (link)]. The primary antibodies used in the analysis are displayed in Table 1. Protein bands were acquired on a Fusion-FX6 imaging system (Vilber Lourmat, Marne-la-Valle, France). GAPDH was used as a loading control.