Anti nlrp3

Western blot analysis using whole-cell lysates and cell culture supernatants was performed as described previously (Hornung et al., 2008 (link); Liu et al., 2009 (link)). The cell culture supernatants (400 μl) were lysed, precipitated by the addition of an equal volume of methanol and 0.25 volumes of chloroform, vortexed and centrifuged for 10 min at 20,000 × g. The upper phase was discarded, and 500 μl of methanol was added to the interphase. This mixture was centrifuged for 10 min at 20,000 × g, and the protein pellet was dried at 55 °C, resuspended in Laemmli buffer and boiled for 5 min at 99 °C. The protein lysates from the HK-2 cells and kidney cortex extracts were lysed using a total protein extraction kit (KeyGEN, China) according to the manufacturer’s instructions, separated by SDS-PAGE, and then transferred onto PVDF membranes (Millipore) blocked with 5% milk proteins. The membranes were then incubated overnight at 4 °C with the following primary antibodies: anti-Nlrp3, IL-1β, IL-18, ASC, caspase-1, MCP-1, IL-6 (Santa Cruz Biotechnology, USA), and IL-1β (Cell Signaling technology, USA). The blots were washed and incubated with secondary horseradish peroxidase-conjugated antibodies as appropriate, and the signals were then detected using an ECL advanced system (GE Healthcare, UK).

Mouse AMϕ were lysed (∼1 × 10⁶ cells/ml) in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na₃VO₄, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 20 mM PMSF). Protein levels were quantified, and 600 μg of total protein for each sample was then immunoprecipitated with anti-ASC antibody (Santa Cruz Biotechnologies, CA), anti-NOD2 antibody (Santa Cruz Biotechnologies, CA), or anti-p62/SQSTM1 antibody (Sigma-Aldrich, St. Louis, MO). The immunoprecipitated proteins were separated on a 10% SDS-PAGE gel, and were then electroblotted onto PVDF membrane and blocked for 1 h at room temperature with Tris-buffered saline containing 3% non-fat dried milk. NLRP3 and RIP2 protein was detected by probing the membranes with anti-NLRP3 and anti RIP2 antibodies (Santa Cruz Biotechnologies, CA) at 1:500 dilution, respectively, and detected with Clean-Blot IP Detection Reagent (Thermo Scientific, Rockford, IL) following the manufacturer's instructions. Blots were then stripped and reprobed with anti-ASC antibody or anti-NOD2 antibody, and detected with Clean-Blot IP Detection Reagent. Caspase-1 cleavage in the AMϕ was measured by detecting its p10 fragment in Western blot using rabbit polyclonal anti-mouse caspase-1 p10 (Santa Cruz Biotechnologies, CA).