Complete protease inhibitors

For Western blot analysis, cells were lysed in 1× lysis buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100; Cell Signaling) supplemented with 1× complete protease inhibitors (Roche). Twenty to 50 μg of total protein was incubated overnight at 4°C with the following antibodies: 5 μg KDM4C antibody (A300-885A, Bethyl Laboratories), 5 μg hnRNPL antibody (ab6106, Abcam), 2 μg beta-tubulin (Millipore), or 2 μg TBP (ab818, Abcam). KDM4C protein levels were quantified using ImageJ software normalized to hnRNPL protein levels and are shown as the fold difference relative to the individual with the lowest expression.

Culture supernatants of DCs treated with MTX and TCL for 2 h were collected and precipitated with equal volumes of methanol and 1/4th volume of chloroform. The solutions were vortexed and centrifuged for 15 min at 20,000 × g, and the upper phase was discarded. After adding 600 μL methanol to the interphase, the mixture was centrifuged for 15 min at 20,000 × g, and the resulting protein pellets were dried at 55°C and re-suspended in loading buffer. The cell pellets were homogenised with a cell lysis buffer containing Phenylmethylsulfonyl fluoride (PMSF), complete protease inhibitors and phosphatase inhibitors (Cell Signaling Technology). The lysates were quantified by BCA assay (Thermo Fisher Scientific), and equal amounts of proteins were mixed with the loading buffer. All protein samples were boiled for 5 min, separated using a 12 % SDS-PAGE gel and then transferred onto nitrocellulose membranes. The blots were incubated with primary antibodies against caspase-1, cleaved caspase-1, IL-1β, cleaved IL-1β, NLRP3, and β-actin at the recommended dilutions, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The bands were visualised using enhanced chemiluminescence reagents from Cell Signaling Technology and Abcam.