Af 201

PBMCs, monocytes or macrophages were lysed in (5×) Laemmli SDS buffer supplemented with 1% β-mercaptoethanol, 0.01% bromophenol blue and protease inhibitor cocktail (Roche). Equal amounts of protein were analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and incubated with primary and secondary antibodies following the manufacturers’ instructions. Primary antibodies against ASC (1:2000, AL177), caspase-1 (1:1000, Bally-1) and NLRP3 (1:1000, Cryo-2) were from Adipogen. Anti-IL-1β (1:500, AF201) antibody was from R&D. Anti-GAPDH and anti-Actin antibodies (1:5000) were from Cell Signaling. Secondary HRP-linked antibodies against rabbit and mouse IgG (1:2000) were from Cell Signaling and against goat IgG (1:2000) was from R&D. Immunoblots were revealed using the enhanced chemiluminescence reagent (Thermo Scientific) and visualized using a BIORAD camera (Universal Hood II). GAPDH or Actin was used as loading controls.

Protein extracts from cell lysates were separated on NuPAGE 4–12% Bis-Tris Protein Gel (NP0301BOX, Invitrogen) according to manufacturer’s instruction. Transfer of separated proteins to nitrocellulose membrane was carried out using iBlot 2 Gel Transfer Device (IB21001, ThermoFisher Scientific). Membranes were blocked in 5% BSA/TBST and stained using human IL-1beta/IL-1F2 (AF-201, R&D systems), ROR alpha (NBP2-24493, NOVUS biologicals), beta tubulin (ab6046, Abcam) and PCNA (FL-261, sc-7907, Santa Cruz Biotechnology) antibodies. Blots were then developed with SuperSignal West Femto maximum sensitivity substrate (34096, Pierce) and imaged with a LI-COR Odyssey Fc system.

To obtain representative cores for the TMA construction, parallel sections stained by hematoxylin and eosin were used to identify a representative core position within the specimens. IHC staining was carried out according to the antibody manufacturers’ instructions. Tissue slides were incubated overnight at 4 °C with a human IL-1 beta/IL-1F2 (AF-201, R&D systems Inc., Minneapolis, MN, USA) and IL-1 RA/IL-1F3 (AF-280, R&D systems, Minneapolis, MN, USA) polyclonal goat immunoglobulin, respectively [12 (link),15 (link)], in dilutions 1:10 and 1:300, in real antibody diluent (DAKO, Glostrup, Denmark). After three more washing steps, a visualization was performed with Dako Liquid DAB-Substrat Chromogen System K3467 (DAKO, Glostrup, Denmark) and counterstaining with hematoxylin, as indicated by the manufacturer [27 (link)]. Two or more cores of every invasive BC and corresponding normal bladder tissue were integrated.