Anti tms1

An equal number of cells were seeded per sample in order to ensure equal sample loading for all supernatants analysed by Western blot. Thus, 750 000 cells per well were seeded when using 6-well plates, or they were seeded to 75–80% confluence in cell-culture flasks. Proteins from cell-free culture supernatants were concentrated using trichloroacetic acid (TCA) (Koontz, 2014 (link)). One hundred percent TCA was added to supernatants for a final concentration of 20%. Samples were vortexed and incubated on ice for 30 min. After incubation, samples were centrifuged at 800 × g and supernatants were discarded. The pellets were washed twice with 100% cold acetone and allowed to air-dry. Pellets were resuspended with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific) and boiled at 95°C for 5 min. Samples were loaded and run on SDS-PAGE gels and transferred onto PVDF membranes. Membranes were blocked with 5% BSA for 1 h at RT, and then incubated with anti-TMS1 (Abcam), caspase-1 (Nathan), IL-1β (Abcam), anti-NLRP3 (Cell Signaling) or HMGB1 (Nathan) primary antibodies overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit HRP (Millipore) for another hour. Finally, membranes were washed and exposed to Luminata Forte (Millipore) substrate. Images were acquired using ChemiDoc XRS+ system and analysed using NIH-ImageJ.