Analyzing Inflammasome Signaling in Neutrophils

A total of 10⁶ neutrophils were lysed and analyzed by Western blotting as previously described (26 (link)) using the following antibodies: anti-IL-1β (3ZD; National Cancer Institute Biological Resources), anti-NLRP3 (D2P5E; Cell Signaling), anti-caspase-1 (Cell Signaling), anti-NF-κB p65 (D14E12; Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1; Cell Signaling), anti-IκBα (L35A5; Cell Signaling), anti-MyD88 (D80F5; Cell Signaling), anti-TRAF6 (D21G3; Cell Signaling), anti-IKKα (Cell Signaling), anti-IKKβ (2C8; Cell Signaling), anti-phospho-IKKα/β (Ser176/180) (16A6; Cell Signaling), anti-CREB1 (48H2; Cell Signaling), anti-phospho-CREB (Ser133) (87G3; Cell Signaling), anti-C/EBPβ (Cell Signaling), anti-phospho-C/EBPβ (Thr235) (Cell Signaling), or anti-β-actin (AC-15; Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were used (BioLegend). Membranes were developed using ECL (Thermo Scientific) and detected using a Nikon camera as previously described (79 (link)). Quantification analysis of blots was performed using ImageJ, and β-actin was used as a loading control. The results for samples were expressed as a percentage of the value for the positive control (LPS or LPS+ATP group).