SDS-PAGE and Western Blot Analysis

Cells or NET-precipitated proteins were lysed on ice in SDS lysis buffer (2% SDS, 62.5 mM Tris at pH 6.8, 5% 2-mercaptoethanol, 10% glycerol) supplemented with Complete Roche Inhibitor Cocktail (Complete tablets Mini Easypack [catalog 04693124001] and PhosSTOP Easypack [catalog 04906837001]) and centrifuged at 13,000g for 15 minutes at 4°C. Whole-cell lysates (20–30 μg protein) were subjected to SDS-PAGE electrophoresis on 12.5% gels and then transferred to an Immobilon-PSQmembrane (catalog SEQ85R; Millipore). Membranes were blocked with 5% skimmed milk in TBS plus Tween 20 and then incubated with anti-MPO (1:3000 dilution; Dako) as loading control (78 (link)) or anti–IL-33 (1:1000 dilution) specific antibodies (mouse “Nessy” antibody; R&D). For HL-60 protein extracts, mouse anti–human tubulin antibody was used as loading control (1:3000 dilution, catalog A11126; Thermo Fisher Scientific). Detection was performed using relevant HRP-linked antibodies (anti–goat-HRP, anti–rabbit-HRP, anti–mouse-HRP; catalog AP180P, 12-348, and 12-349, respectively; all purchased from Millipore) and enhanced chemiluminescent-detection reagents (Amersham Biosciences, RPN2109). Unedited gels are provided in the online supplement.