Anti p65 antibody

ChIP assay was performed with a ChIP Assay Kit (Millipore) according to the manufacturer's instructions with an anti-P65 antibody (2 μg; Millipore), anti-c-Rel antibody (2 μg; Millipore) and normal mouse IgG as an NC. qPCR was performed using Taqman Universal PCR Mix as described above, and the primers used for amplification of the immunoprecipitated DNA are listed in Supplementary Table S1. All experiments were repeated three times.

The ChIP analysis was performed using the Millipore EZ-ChIP kit according to the manufacturer's protocol (Millipore, Temecula, CA, USA). In brief, MCs were cross-linked with 1% formaldehyde for 10 min at room temperature after transferred with siGm4419 or Gm4419 (+) and lysed in SDS lysis buffer with 1 mM PMSF. The chromatin was sheared by sonication to an average size of 200–1000 bp. The part of the total lysate was used as ‘Input DNA' control. The chromatin solutions were immunoprecipitated overnight at 4 °C using anti-p50 antibody (Abcam, 1 mg, Carlsbad, CA, USA), or anti-p65 (Abcam, 1 mg), normal rabbit IgG (Millipore, 1 mg) replaced the anti-p50 antibody or anti-p65 antibody as negative control. Salmon sperm DNA-saturated protein G Sepharose was added to protein/DNA complexes for 2 h at 4 °C. The samples that cross-linked by formaldehyde were reversed in 5 mM NaCl at 65 °C overnight after extensive washing. After DNA purification by phenol/chloroform and precipitated with ethanol, the genomic DNA fragments were analyzed by qRT-PCR (primers corresponding to sequences within the promoter regions of Gm4419 and NLRP3 inflammasome were shown in Supplementary Table 3).