Fig. 5.

The activation of the NLRP3 pathway is dependent on the FHA and RING-like domain of Pellino2. WT and Peli2^−/− BMDMs were infected with GFP-expressing murine stem cell virus (MSCV) as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (Peli2-RING), or Pellino2 FHA mutant (Peli2-FHA). a GFP expression (left panels) and immunoblot analysis of myc (right panel) (scale bar = 20μm). b ELISA of IL-1β and IL-18 in medium from virus-infected WT and Peli2^−/− BMDMs stimulated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 1 h. c, d Immunoblot analysis (c) and densitometry analysis (d) of IL-1β and Caspase-1 in medium (Sup) and cell lysates from virus-infected WT and Peli2^−/− BMDMs stimulated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 1 h. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (paired, two-tailed Student’s t-test (b) or two-way ANOVA (d)). Data are biological replicates that are representative of 3–5 independent experiments (a, c) or represent the mean ± s.e.m. of 3–5 independent experiments (b, d)