Western blot analysis of NLRP3 using anti-NLRP3 antibody (PA1665). Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: rat thymus tissue lysates, Lane 2: mouse thyus tissue lysates, Lane 3: mouse RAW264.7 whole cell lysates, Lane 4: mouse J774A.1 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NLRP3 antigen affinity purified polyclonal antibody (PA1665) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for NLRP3 at approximately 110 kDa. The expected band size for NLRP3 is at 118kDa.