Anti mouse caspase 1 ag 20b 0042

Lipopolysaccharides (LPS, #L2880), adenosine triphosphate (ATP, #A3377), and aluminum potassium sulfate dodecahydrate (Alum, #237086) were purchased from Sigma (St. Louis, MO, USA). COS was bought from Abphyto Biotech Co., Ltd. (Chengdu, China). MitoSOX (#M36008) was obtained from Invitrogen (Carlsbad, CA, USA). MitoTracker (#C1049B) and protein A + G agarose (#P2012) were supplied by Beyotime (Shanghai, China). Anti-NLRP3 (#AG-20B-0014) and anti-mouse caspase-1 (#AG-20B-0042) antibodies were purchased from Adipogen (San Diego, CA, USA). Anti-NEK7 (#ab133514) and anti-GSDMD (#ab209845) antibodies were obtained from Abcam (Cambridge, UK). Anti-mouse IL-1β (#AF-401-NA) antibody was from R&D Systems (Minneapolis, MN, USA). Anti-Flag (#20543-1-AP), anti-HA (#66006-2-Ig), and anti-β-actin (#66009-1-Ig) antibodies were purchased from Proteintech (Rosemont, IL, USA).

Serum-free media (1 mL) from stimulated BMDMs were transferred into fresh tubes and centrifuged for 10 minutes at 12,000 rpm at 4°C; 15 µL of 10% sodium deoxycholate was then added to the supernatants, vortexed, and kept on ice. Trichloroacetic acid (72 µL; Sigma-Aldrich) was subsequently added, and the tubes were kept on ice overnight. After centrifugation for 30 minutes at 12,000 rpm at 4°C, the pellet was washed twice with 500 µL ice-cold acetone. The pellet was dried and boiled with LDS Sample Buffer and β-mercaptoethanol (Sigma-Aldrich). The precipitated protein was resolved by 4% to 12% or by 4% to 20% Invitrogen Novex Tris-Glycine Gels (Thermo Fisher Scientific) and transferred onto Immun-Blot Low Fluorescence PVDF Membrane (1704274; Bio-Rad, Hercules, CA, USA). The transferred membranes were blocked with blocking buffer (927-40000; LI-COR, Lincoln, NE, USA) and incubated with anti-mouse caspase-1 (AG-20B-0042; AdipoGen Life Sciences, San Diego, CA, USA). The signal was visualized with secondary antibodies conjugated with IRDye and the Odyssey CLx Imaging System (LI-COR).