Caspase 1 p20 p10

ROT (Catalog No. R8875), LPS (Catalog No. L6143), and 6‐OHDA (Catalog No. H4381) were obtained from Sigma Chemical (St. Louis, MO, USA). Lysis buffer and the enhanced chemiluminescence (ECL) reagent were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Anti‐tyrosine hydroxylase (TH) (Catalog No. 25859‐1‐AP; rabbit polyclonal), IL‐1β (Catalog No. 16806‐1‐AP; rabbit polyclonal), TNF‐α (Catalog No. 17590‐1‐AP; rabbit polyclonal), NLRP3 (Catalog No. 19771‐1‐AP; rabbit polyclonal), caspase‐1/p20/p10 (Catalog No. 22915‐1‐AP; rabbit polyclonal), and GAPDH (Catalog No. 10494‐1‐AP; rabbit polyclonal) antibodies were bought from Proteintech Group (Chicago, IL, USA). Anti‐ionized calcium binding adaptor molecule‐1 (IBA‐1; Catalog No. ab178847; rabbit monoclonal), Alexa Fluor 594 (Catalog No. ab 150,080, goat anti‐rabbit) and Alexa Fluor 488 (Catalog No. ab 150077, goat anti‐rabbit) antibodies were bought from Abcam (Cambridge, MA, USA). Anti‐ASC (Catalog No. abs155599; rabbit polyclonal), and IL‐18 (Catalog No. abs120003; rabbit polyclonal) antibodies were obtained from Absin (Pudong New District, Shanghai, China). Anti‐NLRP3 (Catalog No. BA3677; Goat Polyclonal) antibody was bought from Boster (Wuhan, China).

After the paraffin tissue sections were deparaffinized, they were placed in citrate buffer solution, heated 20 min and then cooled to room temperature. After being incubated with endogenous peroxidase blocking solution for 10 min, the sections were washed three times with PBS for 10 min each time, 5% BSA was added for 1 h at room temperature, and the following primary antibodies were added and incubated overnight at 4 °C: smooth muscle actin (1:200, Proteintech, Rosemont, IL), vimentin (1:200, Proteintech, Rosemont, IL), NLRP3 (1:100, Proteintech, Rosemont, IL), caspase 1/P20/P10, (1:200, Proteintech, Rosemont, IL), and IL-1 beta (1:200, Abcam, Cambridge, UK). The sections were washed three times with PBS for 10 min each time, and the reaction-enhancing solution was added. An appropriate amount of peroxidase-labeled goat anti-rabbit IgG was added and incubated at room temperature for 20 min. After being washed three times with PBS for 10 min each time, DAB was added to the sample for several seconds. After observing the color change under the light microscope, the reaction was terminated by placing the sample in deionized water. The nuclei were stained with hematoxylin, and the slides were examined under the light microscope.

First, equal amounts of protein were leaded in each lane of a polyacrylamide gel. Next, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C. The membrane was then washed and incubated for 2 h at room temperature with horseradish peroxidase-conjugated IgG. Finally, we employed ECL Western blotting detection reagents (Thermo Fisher Scientific) to visualize the protein bands and used Un-Scan-It 6.1 software (Silk Scientific, Orem, UT, USA) to measure the band density.