Imagequantlas4000 chemiluminescence imaging system

Liver proteins were homogenized and then collected by using RIPA lysis buffer. Cytoplasmic and nuclear proteins were isolated using nuclear and cytoplasmic protein extraction kits (Beyotime, China; cat: P0028), according to the manufacturer's instructions. The BCA protein assay reagent kit was used to determine the concentration of total liver protein and the extracted nuclear protein and cytoplasmic protein. An equal amount of protein (30 μg) was separated by 8–12% SDS-PAGE and transferred into PVDF membranes. Next, membranes were incubated with Tris-buffered saline, containing 5% non-fat dry milk for blocking purposes at room temperature for 1 hour. Then, membranes were incubated overnight at 4°C with primary antibodies directed against HMGB1, TLR-4, caspase-3, caspase-9, Bax, Bcl-2, NF- kB p65, iNOS, and COX-2. After washing with TBST, the membrane was incubated with a secondary antibody for 1 h at room temperature. Finally, the reaction was detected with an enhanced chemiluminescent reagent (NCM Biotech, China; cat: P10100). An ImageQuantLAS4000 chemiluminescence imaging system was used to visualize the target proteins (GE Co., USA), and densitometry was performed using the Image J software version 1.80.

The midbrain, the liver, and the BMDM cells protein lysates were fractionated with a RIPA lysis buffer. The protein was electrophoresed through a 10–15% SDS-polyacrylamide gel and blotted through the PVDF-membrane. The membranes were probed with the following primary antibodies: rabbit anti-Caspase-1/pro-caspase-1 (1:500, Millipore, USA), mouse anti-IL-1β/pro-IL-1β (1:1000, Sigma, USA), mouse anti-NLRP3 (1:1000, Adipogen, USA), rabbit anti-NLRP1 (1:5000, Cell Signaling, USA), rabbit anti-NLRP2 (1:1000, abcam, USA), goat anti-NLRC4 (1:2000, Santa Cruze, USA), rabbit anti-AIM2 (1:5000, Santa Cruze, USA), and mouse-β-actin (1:1000, Sigma, USA). The blots were incubated secondary antibodies and the signals were detected by the enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA). The membranes were analyzed using an Image Quant LAS 4000 Chemiluminescence Imaging System (GE Healthcare, USA).

For protein analysis, total proteins were extracted using RIPA buffer and 1% NP-40 lysis buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The extracted proteins were quantified using the Bradford assay and subjected to immunoblot analysis. Briefly, the proteins were separated using SDS-PAGE and transferred to a PVDF membrane (Amersham Bioscience, Uppsala, Sweden). The first antibody, anti-human PARP (#9542s; Cell Signalling), Caspase-3 (#9665s, Cell Signalling), Caspase-9 (#9508s; Cell Signalling), Bax (NB100-56095; Novusbio), Bcl-2 (NB100-56098; Novusbio), PDHA (sc-377092; Santa Cruz), phospho-PDHA (ab177461; Abcam), LDHA (ab84716; Abcam), PDK1 (ADI-KAP-PK112; Enzo), PDK3 (ab182574, Abcam), and GAPDH (sc-32233; Santa Cruz). HRP-conjugated anti-rabbit IgG or anti-rat IgG (all from Invitrogen) were used as secondary antibodies. The specific bands were developed using a western blot detection kit (Bio-Rad, Hercules, CA, United States) using the ImageQuant LAS 4000 chemiluminescence imaging system (GE Healthcare, Munich, Germany).