Western blot analysis
LYSED, SECONDARY, LYSIS
The activity of NLRP3 inflammaosme was detected by Western Blot analysis with whole-cell lysates and cell supernatants. Cells were washed with 1xPBS buffer and then lysed in RIPA lysis buffer with protease inhibitor (1 μg/ml) and incubated on ice for 20 min. Lysates were then centrifuged at 15,000 rpm for 15 min at 4°C. The concentration of protein supernatant was determined using the BCA protein kit. Total cell lysates were resolved by SDS-PAGE and analyzed for NLRP3, pro-caspase-1 and IL-1β protein levels. To examine secreted IL-1β and caspase-1, culture medium was collected, equal volume of methanol and 1/4 volume of chloroform was added and vortex. The mixtures then were centrifuged at 12000 rpm at 4°C for 10 min, the upper was discarded, the bottom was transferred to a new tube and equal volume of methanol was added and vortex. The mixtures were centrifuged at 12000 rpm at 4°C for 10 min, the supernatant was discarded and the pellet was dried for 5 min. 1xloading buffer was added to the pellet and then boiled at 95°C for 5 min. The cell culture medium lysates were resolved by SDS-PAGE and analyzed for cleaved caspase-1 and cleaved-IL-1β protein levels. 20-40 μg total lysate was separated by SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% non-fat milk and then incubated with primary and secondary antibodies respectively. The primary antibodies used in this study were NLRP3 antibody (abclonal A12694, 1:2000), caspase-1 antibody (Proteintech 22915-1-AP, 1:2000), IL-1β (abclonal A1112, 1:2000), and GAPDH antibody (Proteintech 60004-1-Ig, 1:50000). Western HRP Substrate (Millipore) was used for development of protein bands.