Nf κb p65

The levels of phosphorylated (p)-STAT1 (rabbit polyclonal antibody, dilution 1:1,000, CST, Boston, USA), p-STAT3 (rabbit monoclonal antibody, dilution 1:1,000, CST, Boston, USA), p-STAT4 (rabbit polyclonal antibody, dilution 1:500, Abcam, Cambridge, UK), NF-κB P65 (rabbit polyclonal antibody, dilution 1:4,000, Abcam, Cambridge, UK), p-P65 (rabbit polyclonal antibody, dilution 1:500, Abcam, Cambridge, UK) in the brain of mice were detected by western blot. Firstly, the brains of mice were collected and snap frozen at −80°C immediately. Next, the brain homogenates were prepared in lysis buffer (P0013) consisting of 1 nM PMSF and phosphatase inhibitor. Proteins were denatured and equal amounts of proteins were electrophoresed in 8% bis-Tris/polyacrylamide gels (P0012A; Beyotime, shanghai, China) and transferred to PVDF membranes with 0.45 μm pores (Millipore, Boston, MA, USA). The membranes were blocked for 2 h in blocking solution (TBS containing 5% nonfat dry milk and 0.1% Tween 20) and incubated overnight at 4°C with primary antibody diluted in blocking solution, followed by incubation with horseradish peroxidase-conjugated secondary antibody at room temperature for another 2 h. Last, the immunoreactivity was detected with enhanced chemiluminescence (PPLYGEN).

The RIPA buffer (Beyotime, China) was utilized to extract protein samples. Then, the concentration of these protein samples was determined with the BCA kits (Beyotime, China). Next, these proteins underwent the separating procedure in 10% SDS-PAGE gel. After that, these proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The PVDF membranes were blocked with 5% skimmed milk at room temperature for 2 h and incubated with primary antibodies at 4°C overnight. The primary antibodies used were Ki-67 (Abcam, ab16667), HMGB1 (Abcam, ab18256), RAGE (Abcam, ab216329), Bcl-2 (Abcam, ab32124), Bax (Abcam, ab32503), Cleaved caspase3 (Abcam, ab32351), cyclin D1 (Abcam, ab16663), TLR4 (Abcam, ab13556), NF-κB p65 (Abcam, ab207297), NF-κB (Abcam, ab281518), caspase4 (Abcam, ab25898), NLRP3 (Abcam, ab263899) and GAPDH (Abcam, ab8245). On the following day, the membranes were incubated with peroxidase-conjugated secondary antibody. Finally, the immunoreactive signals were detected with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA).

Total protein was extracted from cells and liver tissues using RIPA lysis buffer and phenylmethylsulfonyl fluoride (Beyotime, China). The protein concentration was detected by using a BCA protein assay kit. Equal amounts of protein (20 μg) were separated using 10% or 12% SDS-PAGE and were transferred onto polyvinylidene difluoride membranes (PVDF). Next, the PVDF membranes were blocked with 5% fat-free milk and incubated with primary antibodies to NLRP3 (Abcam, Inc., CA, USA; Cat. No. ab-270449), NF-κB/p-65 (Abcam, Inc., CA, USA; Cat. No. ab-76302), and GAPDH (Abcam, Inc., CA, USA; Cat. No.ab-181602) overnight at 4 °C. Subsequently, the membranes were washed and incubated with secondary antibodies at room temperature. The optical density of the bands was visualized by an ECL system (Pierce). GAPDH was used as an endogenous control. Data was normalized to GAPDH levels.

Cell lysis was performed with RLN-T buffer (RLN buffer plus 0.5% Triton X-100 and the complete protease inhibitors cocktail EDTA-free; Roche) at 20 × 10⁶ cells/ml for 5 min on ice. Extracts were centrifuged at 11,000 × g for 15 min at 4°C. The primary antibodies used were as follows: anti-IF1 (1:200) (22 (link)), e-cadherin (1:250, BD Biosciences), β1 integrin (1:2, kindly provided by Carlos Cabañas, CBMSO), β-catenin (1:500, BD Biosciences), vimentin (1:1,000, Cell Signaling), NF-κB p65 (1:1,000, Abcam), pIKBα (1:500, Cell Signaling), IKBα (1:1,000, Cell Signaling), α-tubulin (1:3,000, Sigma-Aldrich), β-F1-ATPase (1:25,000) and Hsp60 (1:2,000) from Ref. (35 (link)), SDH-B (1:500, Invitrogen),α-F1-ATPase (1:1,000, Molecular Probes), Core 2 of complex III (1:500, Abcam), MTCO2 (1:500, Abcam), VDAC (1:500, Abcam), and PYGM (1:1,000, Abcam).