Ag 20b 0042 c100

Rabbit monoclonal Anti-ASC (AG-25B-006-C100, AdipoGen, San Diego, CA, USA), mouse monoclonal anti-NLRP3 (AG-20B-0014-C100), rabbit monoclonal anti-caspase-1 (p20) (AG-20B-0042-C100), Rabbit monoclonal anti-GSDMD (ab209845, ABCAM, Cambridge, UK), rabbit monoclonal anti-PBR (ab109497, ABCAM), mouse Antiβ-actin (SJ190a9b68548, Sigma-Aldrich, St. Louis, MO, USA), rabbit monoclonal anti-IL-1β (12242, CST, Danvers, MA, USA), rabbit monoclonal anti-Iba1 (PA5-21274, Invitrogen), mouse monoclonal anti-Iba1 (019-19741, WAKO, Osaka Japan), anti-cytochrome C antibody (ab90529, Abcam) FITC anti-mouse CD45 antibody (103108, BioLegend, San Diego, CA, USA), APC anti-mouse F4/80 antibody (123116, BioLegend), PE/Cyanine7 anti-mouse CD86 antibody (105014, BioLegend), PE anti-mouse/human CD11b antibody (101208, BioLegend).

Protein was extracted using RIPA buffer (ThermoFisher). Lysates were added with SDS sample loading buffer and dithiothreitol (DTT). Denatured samples were separated via SDS-PAGE and were transferred to nitrocellulose membranes. The membranes were blocked using fresh 2% nonfat milk in 1× tris buffered saline with Tween 20 (Santa Cruz, sc-362311) at room temperature for 1 h before incubation with antibodies at 4°C overnight. After washing, the membranes were incubated with secondary antibodies at room temperature for 1 h and were visualized using an Odyssey Imaging System (LICOR) or X-ray films. Antibodies: mouse caspase-1 (Adipogen, AG-20-B-0042-C100), mouse GSDMD (Abcam, ab209845), mouse IL-1β (R&D, AF-401-NA), full-length mouse caspase-8 (Cell Signaling Technology, CST, 1C12), cleaved mouse caspase-8 (CST, D5B2), mouse/human β-actin (Sigma, AC-15), mouse/human GAPDH (Sigma, GAPDH-71.1), IRdye 800C goat anti-rabbit IgG (LICOR, 926-32211), IRDye 680RD goat anti-mouse IgG (LICOR, 926-68070), goat anti-rabbit IgG HRP (Bio-Rad, 1705046), goat anti-mouse IgG HRP (Bio-Rad, 1721011).