Asc antibody

To assess the protein composition and association of proteins in the inflammasome. RPMI cultures were lysed in 1 ml of Radio Immunoprecipitation Assay (RIPA) [1% NP‐40, 0.5% DOC (Deoxycholic acid; Sigma D6750; Sigma‐Aldrich), 0.1% SDS, 50 mM Tris.HCl, pH 8.0, 150 mM NaCl] with protease inhibitor mixture (Sigma‐Aldrich). About 2 mg of cell lysates were immunoprecipitated with 4 μg ASC‐antibody (rabbit monoclonal; Santa Cruz Biotechnology) and 50 μl protein G‐Agarose (p4691; Sigma‐Aldrich) in 4°C for 16 hrs with nutation, and then spin down the beads at 2.4 × g for 30 min. at 4°C. The pelleted beads were washed four times with PBS buffer (pH 7.3, 137 mM NaCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). After the last wash, spin down once more at 5000 r.p.m. for 5 min. at 4°C without adding any buffer followed by aspirating the residual buffer. Add 15–20 μl SDS loading buffer mix the buffer and bead with finger tickling, and then boil the mixture for 10 min. and spin down with 14,000 r.p.m. for 10–30 sec. before analysis by Western Blotting using antibodies against NALP3 (mouse monoclonal, 1:400 dilution; Adipogen), pro‐caspase‐1 (mouse monoclonal, 1:400 dilution; Santa Cruz Biotechnology), and ASC (rabbit monoclonal, 1:400 dilution; Santa Cruz Biotechnology).

The following reagents were purchased: docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA, Cayman Chemical); Poly(dA:dT), adenosine triphosphate (ATP), and nigericin (Sigma-Aldrich); Lipopolysaccharide (LPS, ENZO Life Sciences); purified flagellin (InvivoGen); neutralizing antibody to IL-1β (R&D Systems); caspase-1 antibody (Santa Cruz Biotechnology); NLRP3 antibody (ENZO Life Sciences); ASC antibody (Santa Cruz Biotechnology); and the appropriate secondary antibodies (Jackson ImmunoResearch Laboratory). For the ImageStream analysis a primary rabbit polyclonal NF-κB/p65 antibody (SantaCruz Biotechnology) was used with an Alexa647 conjugated donkey anti rabbit IgG antibody (Jackson ImmunoResearch Laboratories). DNA was stained using DAPI (20 µM).

Deoxycholic acid (DCA), lipopolysaccharide (LPS), CA-074 Me, and JTE-013 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextran sulfate sodium (DSS, molecular weight 36–50 kDa) was obtained from MP Biomedicals Inc. (Irvine, CA, USA). ASC antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IL-1β, ERK, AKT antibodies, and U0126 were purchased from Cell Signaling Technologies (Beverly, MA, USA). DMEM and RPMI 1640 were obtained from Invitrogen (Carlsbad, CA, USA). ELISA Kits were from eBioscience (San Diego, CA, USA).