α nlrc4

THP-1 and HEK293T cells were lysed with RIPA buffer supplemented with cOmplete protease inhibitors (11697498001; Roche Biochemicals, Mannheim, Germany). Whole-cell lysates were incubated on ice for 30 minutes and clarified by means of centrifugation. Whole-cell lysates were eluted with SDS-PAGE sample buffer, resolved on Novex 4-12% SDS-PAGE gels with MES running buffer, and subsequently transferred onto nitrocellulose membranes. Membranes were blocked overnight in 3% BSA plus 0.1% Tris-buffered saline–Tween 20 at room temperature for 1 hour and then probed overnight at 4°C with primary antibodies, including α-NLRC4 (rabbit α-NLRC4; D5Y8E; Cell Signaling Technology, Danvers, Mass), α–caspase-1 (mouse α–caspase-1 p20; AdipoGen, San Diego, Calif), α-ASC (rabbit α-ASC; sc-22514; Santa Cruz Biotechnology, Dallas, Tex), α–caspase-8 (mouse α–caspase-8; #9746; Cell Signaling Technology), and α-actin (goat α-actin; sc-1616; Santa Cruz Biotechnology).

Cells were lysed in 1% NP40 buffer (1% NP40, 10% glycerol, 20 mM Tris–HCl (pH7.5), 150 mM NaCl, 1 mM EGTA) complemented with complete protease inhibitors (#11697498001, Roche Biochemicals, Basel, Switzerland) and 1 mM PMSF (Roth, Karlsruhe, Germany) and incubated on ice for 30 min. The lysates were clarified by centrifugation for 15 min at top speed and eluted in SDS-PAGE sample buffer. SDS-PAGE was performed using Novex 4–12% gels (Life Technologies, Carlsbad, CA) with MES running buffer (Thermo Fisher Scientific, Waltham, MA) and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Kenilworth, NJ). The membranes were blocked in 5% skim milk plus Tris-buffered-saline-Tween 20 (TBST) at room temperature for 1 h and then incubated in primary antibody overnight at 4 °C. Primary antibodies used are α-NLRC4 (rabbit anti-human NLRC4, D5Y8E, Cell Signalling Technologies, Danvers, MA), β-Actin-HRP (C4) (sc-47778, Santa Cruz Biotechnology, Dallas, TX), and monoclonal anti-FLAG M2-peroxidase (HRP) antibody (Sigma-Aldrich, St. Louis, MO).