Al177

Ultrapure lipopolysaccharide (LPS) (E. coli 0111:B4, cat. no. tlrl-3pelps), standard LPS (E. coli 0111:B4, cat. no. tlrl-eblps), ATP (cat. no. tlrl-atpl), nigericin (cat. no. tlrl-nig), and MSU (cat. no. tlrl-msu) were purchased from InvivoGen, lipofectamine 3,000 transfection reagent (cat. no. L3000015) was purchased from Thermo Fisher, C646 (S7152) was bought from Selleck Chemicals, mouse immunoglobin IgG protein (cat. no. ab198772) was purchased from Abcam, Protein A/G PLUS-Agarose (cat. no. sc-2003) was obtained from Santa Cruz, cell lysis buffer (CLB) (cat. no. 9803) was purchased from Cell Signaling Technology, and mouse IL-1β (cat. no. 88–7013), tumor necrosis factor-α (TNF-α) (cat. no. 88-7324), interleukin-6 (IL-6) (cat. no. 88-701364), and human IL-1β (cat. no. BMS22) ELISA kits were purchased from Thermo Fisher.
Anti–IL-1β (1:1,000, AF-401-NA; RRID:AB_416,684) was purchased from RD System, anti-NLRP3 (1:1,000, Cryo-2) and ASC (1:1,000, AL177) were purchased from Adipogen, anti–caspase-1 (1:1,000, ab179515), and anti-NEK7 (1:5,000, ab133514) were purchased from Abcam; Anti–β-actin (1:10,000, BH10D10), anti-p65 antibody (1:1,000, 8242), anti–p-p65 antibody (1:1,000, 3033), and GAPDH antibody (1:2,000, 5,174) were purchased from Cell Signaling Technology, and DyLight 488–labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen.

Protein lysates from splenic cells were harvested by RIPA buffer as previously described (10 (link)), heat denatured in sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) and resolved through 10-15% SDS-polyacrylamide gel by electrophoresis, then transferred to Immobilon-FL membrane (Millipore). Membranes were treated with blocking buffer (LI-COR) then probed with primary and secondary antibodies conjugated with fluorophore, and visualized and quantified using the Odyssey Imaging System (LI-COR). Lysates were probed with anti-IL1β, -IL18, -CASP1-p10 (sc-514 Santa Cruz Biotechnology Inc.), anti-apoptosis-associated speck-like protein containing a CARD (ASC) and -NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) antibodies (AL177 and Cryo-2, respectively, Adipogen), -eukaryotic initiation factor 2 (Eif2) and -phospho-Eif2 Ser51) (#9722 and 119A11, respectively, Cell Signaling Technology) and protein loading was assessed with anti-β-actin (ab8226 Abcam) or -α-tubulin (3873 Cell Signaling) antibodies.

Primary peritoneal macrophages were seeded in chamber slides and the following day, the cells were given with indicated stimuli. Then we used 4% Paraformaldehyde (PFA) to fix the cells for 15 min at room temperature. Then the cells were washed with PBS, permeabilized with 0.1% Triton X-100, and blocked with PBS buffer which were containing 3% BSA. Then we used the anti-ASC (Adipogen AL177, 1:200 at 4°C overnight) to incubate with the cells and used DyLight 488-labeled as the secondary antibody (1:50 at room temperature for 45 min). Finally, DAPI was used to stain nuclei and cells were visualized by fluorescence microscope (Nikon Ti2-U).