Detecting Pro-IL-1β and Processed IL-1β

Pro-IL-1β and processed IL-1β forms were detected by immunoblotting, as previously described [17 (link), 50 (link), 54 (link)]. Briefly, supernatants were precipitated with 10% trichloroacetic acid (Sigma-Aldrich) and washed in cold acetone by centrifugation at 12,000 × g for 10 min at 4°C. Next, pellets were dried and resuspended in sample buffer. For analysis of cell lysates, the plates were incubated on ice for 10 min, and then the cells were detached with a scraper and transferred into 1.5-ml Eppendorf tubes. Cells were lysed with 50 μl of lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1% Triton X-100, and 5% glycerol, all purchased from Sigma-Aldrich) supplemented with a mixture of protease inhibitors (Roche). The lysates were centrifuged at 12,000 × g for 10 min at 4°C, to eliminate cellular debris and proteins were separated on 15% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Immunoblotting was performed using goat anti-IL-1β (1:800; R&D Systems). Immunoblots were developed using HRP-conjugated secondary antibodies (1:15,000 R&D Systems) and developed with Luminata Forte Western HRP substrate (Biorad).