Ab32518

A PierceTM BCA Protein Assay Kit, RPMI1640 medium, DPBS/MODIFIED, Trypsin-EDTA Solution, fetal bovine serum, and dual antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). A CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) kit was obtained from Promega (Madison, WI, USA) and phenazine methosulfate was purchased from Sigma-Aldrich (St. Louis, MO, USA). High Efficiency RIPA tissue/cell rapid lysis solution was acquired from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All antibodies were purchased from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Beverly, MA, USA) as follows: Nuclear Factor KappaB (NF-κB) p65 (D14E12) XP Rabbit mAb (#8242, dilution 1/1000), p-NF-κB p65 (Ser536) (93H1) rabbit mAb (#3033, dilution 1/1000), anti-IκBα antibody (ab32518, dilution 1/1000), anti-SDHA antibody (ab137040, dilution 1/1000), and anti-Lamin B antibody (ab133741, dilution 1/1000).

Proteins were extracted from the PVN samples by protein lysates, and the protein concentrations were measured using the BSA assay. Equal amounts of protein samples were separated by a 10%–12.5% gel and incubated with TLR4 antibody (GeneTex, GTX64330, 1:1000), anti‐MyD88 antibody (Abcam, ab219413, 1:1000), anti‐NF‐kB p65 (Abcam, ab16502, 1:1000), anti‐IkB alpha antibody (Abcam, ab32518, 1:1000), GAPDH (Cell Signaling, #2118, 1:1000) and anti‐histone antibody (Bios, bs‐17422R, 1:1000) overnight at 4°C. Then, they were incubated with HRP goat anti‐mouse IgG (H + L) (ABclonal, AS003, 1:2000) and goat anti‐rabbit IgG‐HRP (Absin Bioscience Inc., abs20040ss, 1:5000) for 1 h at room temperature on a shaker. The protein strips were developed with Stain‐Free Western blotting technology (Bio‐Rad) and analysed with ImageJ software (Bio‐Rad, v.1.8.0.112).

Protein was extracted using a RIPA lysis buffer including a protease inhibitor combination (Sigma-Aldrich) that had been freshly added. Supernatants were collected after centrifugation. Bicinchoninc acid reagent (Thermo Scientific) was applied to assess protein concentration. The cytoplasmic and nuclear levels of NF-κBp65 were examined using an NE-PER kit (Thermo Scientific). Sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (Millipore, Bedford, USA) were used to isolate the proteins and then transferred onto nitrocellulose membranes. After blocking in 5% non-fat dry milk, the membranes were then incubated overnight with the following antibodies: TRIM64 (Novus Biologicals, LLC, Centennial, CO, USA; NBP2-83,713), CD36 (Abcam; ab133625), ABCA1 (Abcam; ab18180), NF-κB (Abcam; ab16502), NLRP3 (Abcam; ab232401), ASC (Abcam; ab155970), caspase-1 (Abcam; ab207802), GSDMD-N (Abcam; ab215203), anti-IκBα (Abcam; ab32518), H3 (Cell Signaling Technology; 4499), and GAPDH (Cell Signaling Technology; 5174) followed by the horseradish peroxidase-conjugated secondary antibody. Immunoreactive band was visualized with enhanced chemiluminescence detection kit reagents (Servicebio, Wuhan, China).