Immunoblotting
1×106 cells were seeded in 12-well plates, stimulated as indicated, and lysed in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentrations were determined by Bradford assay (Thermo Scientific). Lysates were resolved by electrophoreses on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). Blots were blocked for 1 hour at room temperature and probed overnight at 4°C for pro-IL-1β (Santa Cruz), IκBα (Cell Signaling), or β-actin (Cell Signaling). Blots were subsequently probed with fluorescently-labeled secondary antibodies, IRDye® 680 or 800CW (LI-COR Biosciences) for 1 hour. Both blocking and probing steps were carried out in tris-buffered saline (G Biosciences) containing 5% bovine serum albumin and 0.1% TWEEN 20 (Calbiochem). Blots were imaged on a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantified using the included analysis software.