NLRP3 Inflammasome Pathway Activation

Cell lysates were collected by centrifugation (10,000× g) at 4 °C for 30 min. After centrifugation, the proteins in the supernatant were subjected to separation by sodium dodecylsulfate polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride membrane. The membrane was immersed in 5% non-fat milk for 1 h prior to incubation with the primary antibodies at 4 °C overnight. Primary antibodies against GAPDH (1:5000), NLRP3 (1:1000), caspase-1 (1:1000), pro-caspase-1 (1:1000), ASC (1:1000), p-NF-κB (1:1000), NF-κB (1:1000), IL-1β (1:1000), p-ERK1/2 (1:1000), ERK1/2 (1:1000), p-JNK (1:1000), and JNK (1:1000) were used in the immunological detection. Membranes were rinsed with Tris-buffered saline containing 0.1% Tween^® 20 (TBST) 3 times prior to incubation with secondary antibodies at 37 °C for 1 h. Then, the target protein bands were imaged using the enhanced chemiluminescent reagent (Invitrogen) and quantitated by the iBright Imaging Systems (iBright FL 1000; Thermo Fisher Scientific, Inc., Waltham, MA, USA).