Sc 393581

After choosing RIPA buffer (Solarbio, R0010) to lyse cells, we then centrifuged the turbid liquid and collected the supernatant. Protein samples were quantified to 20 μg by a BCA protein assay kit (Solarbio, PC0020), then loaded in SDS-PAGE for further separation and later transferred onto PVDF membranes. The PVDF membranes were blocked with 5% fat-free milk at room temperature for 1 h. After being washed 3 times, the membranes were soaked in primary antibodies against GSDMD (Santa Cruz, sc-393581, 1 : 200), MyHC (Abcam, ab50967, 1 : 1000), NLRP3 (Abcam, ab263899, 1 : 1000), IL-1β (Abcam, ab283818, 1 : 1000), and IL-18 (Abcam, ab191860, 1 : 1000) at 4°C overnight. The membranes were subsequently reprobed with GAPDH (Cell Signaling, 2118S, 1 : 1000) for standardization. After being washed three times, they were incubated in anti-mouse IG-HRP (Beyotime, A0216) or anti-rabbit IG-HRP-(Beyotime, A0208) secondary antibodies for 1 h. Finally, visualizations of proteins by High-sig ECL Substrate (Bio-Rad, USA) were analyzed with ImageJ software.

Proteins were drawn from the tissues and cells and quantified by using a BCA protein kit (Thermo Scientific). Proteins (50 μg) were loaded on SDS-PAGE gels per lane and transferred to PVDF membranes after electrophoresis. Then blocking membranes with 5% BSA at 2 h room temperature and incubating at 4°C overnight with the primary antibodies: GAPDH (Abcam, ab8245, 1:5,000), GSDMD (Santa Cruz Biotechnology, sc-393581, 1:1,000), caspase-11 (Abcam, ab22684, 1:1,000), caspase-1 (Abcam, ab138483, 1:1,000), caspase1 (p10), and caspase-1 (p20) (AdipoGen, AG-20B-0044, AG-20B-0042, 1:1,000). Immunoreactivity was detected by incubating with secondary antibodies (Abcam ab205718, ab97023, 1: 20,000).

Paraffin-embedded sections of the mouse aorta were taken, dewaxed, dehydrated, and subjected to antigen retrieval. Sections were added with 0.03% Triton for 10 min and sealed with normal goat serum blocking solution (C-0005, HaoranBio, Shanghai, China) for 60 min at room temperature. For macrophages, macrophages were fixed with 4% paraformaldehyde for 30 min, washed with precooled PBS And sealed as the above. Next, primary antibodies of HMGB1 (66525-1-Ig, 1:250, Mouse, Proteintech, VA, USA), F4/80 (ab6640, 1:50, Rabbit, Abcam), GSDMD (sc-393581, 1:200, Mouse, SANTA, UC, USA), iNOS (ab210823, 1: 100, Mouse, Abcam), and CD206 (60143-1-Ig, 1:200, Mouse, Proteintech) were selected for sample incubation at 4 ℃ overnight. Then, the samples were incubated with fluorescent secondary antibody, Alexa Fluor^® 647-Anti-Rabbit IgG (ab150075, 1:500, Donkey, Abcam) or Alexa Fluor^® 488-Anti-Mouse IgG (ab150113, 1:500, Goat, Abcam) at room temperature for 60 min in the dark. A fluorescence microscope was utilized for observation, with fluorescence intensity recorded.