Anti irf4

Mouse CD43⁻ resting B cells, which were stimulated with 1 μg/ml anti-mouse IgM Ab and 100 μg/ml poly(I:C) for 72 h, were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Nonidet P-40) in the presence of a protease inhibitor cocktail (Sigma-Aldrich). After 30 min incubation, the cells were sonicated by ultrasonication (Emerson Electric, St. Louis, MO, USA). Supernatants were collected after centrifugation at 15,000 g for 30 min and mixed with SDS sample buffer with 2-mercaptoethanol (Sigma-Aldrich). After 5 min boiling, the proteins were separated by NuPAGE 4–12% Bis-Tris gel electrophoresis (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore). Anti-IRF4 (1:1,000 dilution, BioLegend), anti-IRF5 (1:1,000 dilution, Abcam, Cambridge, MA, USA), anti-IRF8 (1:1,000 dilution, Abcam), anti-ubiquitin (1:2,000 dilution, BioLegend), and anti-β-Actin antibodies (1:4,000 dilution, BioLegend) were used to detect the specific proteins. Chemiluminescence was developed using ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and exposed to the LAS-3000 Mini Imaging System (FUJIFILM, Tokyo, Japan).

ChIP assays were performed with 3 × 10⁶ HTB-176 cells. First, the cells were cross-linked with 1% formaldehyde and then quenched by adding 125 mM glycine at RT. The cells were then lysed, and chromatin was sheared into 200- to 500-bp fragments using a sonicator (SONICS Vibra-Cell, CO, USA). ChIP was performed using the following antibodies: 4 μg anti-NRLP3 (R&D Systems, MI, USA), 4 μg anti-IRF4 (BioLegend Cat# 646411, RRID:AB_2728477), 4 μg of anti-IgG (Thermo Fisher Scientific Cat# 13-4724-85, RRID:AB_470090) or 4 μg anti-CTCF (Millipore Cat# 07-729, RRID:AB_441965). Immunoprecipitated DNA was analyzed by PCR using the primers listed in Supplementary Table S4. An anti-CTCF antibody and the promoter region of the IGF2 gene that harbors a CTCF binding site were used as positive controls. This antibody was also used as a negative control for the NRLP3-specific interaction with the IL-4 promoter.