Anti nlrp3

The total protein was extracted from the liver tissues using a Total Protein Extraction kit (Beyotime, Shanghai, China) and quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein per sample (30 μg) were separated on a 10% SDS–PAGE and electroblotted onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk for 2 h, the blots were incubated overnight with anti-VEGF (1:2000; Wanleibio, Shenyang, Liaoning, China), anti-Cyclin D1 (1:1000; Wanleibio, Shenyang, Liaoning, China), anti-NLRP3 (1:2000; Bioss, Beijing, China), anti-Wnt2 (1:1000; Abcam, Waltham, MA, USA), anti-β-catenin (1:10,000; Abcam, Waltham, MA, USA), anti-P-P65 (1:500; Santa, Dallas, TX, USA), anti-P65 (1:3000; Proteintech, Wuhan, Hubei, China), anti-caspase-1 (1:500; Santa, Dallas, TX, USA), anti-ASC (1:500; Santa, Dallas, TX, USA), anti-GSDMD (1:2000; Affinity, Liyang, Jiangsu, China), and anti-β-tubulin (1:1000; Wanleibio, Shenyang, Liaoning, China) primary antibodies at 4 ℃. The blots were washed with TBST and incubated with HRP-conjugated anti-IgG for 2 h. The positive bands were detected using an enhanced ECL reagent (Meilunbio, Dalian, Liaoning, China) on an AI600 System (GE Healthcare, Pollards Wood, UK). The relative protein expression was quantified using ImageJ software and normalized against the β-tubulin control.

To investigate the colocalization of RACK1, NLRP3 and NEK7, cells were prepared as described above and transfected with the pcDNA3.1-eGFP-NEK7 plasmid for 48 h. Then, the cells were infected with PmCQ2 for 3 or 4 h. After infection, the cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 30 min at room temperature (RT). After three wash steps, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at RT. After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight. Next, secondary Abs including goat anti-mouse IgG (H&L) Alexa Fluor 488 (Abcam, UK), goat anti-rabbit IgG (H&L) Alexa Fluor 594 (Abcam, UK) and ABflo™ 647-conjugated goat anti-rabbit IgG (H&L) (Abclonal, China) were added after washing with PBS and incubated for 1 h at RT. Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added and incubated in the dark for 5 min. Finally, anti-fluorescence attenuation mounting tablets (Solarbio, Beijing, China) were used, and the results were observed using fluorescence microscopy (Olympus, Tokyo, Japan).

Cells were prepared in 48-well plates and treated with LPS and CATH-2 as described above. After 6 h incubation, cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 20 min at room temperature (RT). After three wash steps, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Subsequently, cells were blocked with 5% Bovine Serum Albumin (BSA) in PBS for 30 min. Then, cells were stained with primary antibody containing anti-NLRP3 (Bioss, Beijing, China) and anti-ASC (Santa cruz, CA, USA) for 1 h at RT. After the wash steps, cells were incubated with Goat anti-mouse IgG (H&L) Alexa fluor 488 and Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, UK) for 1 h. DAPI (Beyotime Biotechnology, Shanghai, China) was added for 5 min to visualize cell nuclei. Finally, cells were washed and maintained in antifading medium (Solarbio, Beijing, China). Cells were observed using the fluorescence microscopy (Olympus, Tokyo, Japan).