Fig. 4: MafB −/− macrophages demonstrate enhanced activation of the NLRP3 inflammasome.

a–d Primed MafB+/+ and MafB−/− PMs (a, b) and BMDMs (c, d) were washed and stimulated with or without NLRP3 agonists ATP (5 mM) or Nigericin (5 μM) for 1 h. Levels of the supernatant IL-1β were determined by ELISA (a, c), and levels of the indicated proteins in the cells were determined by western blotting (b, d). e MafB+/+ and MafB−/− PMs were treated as in (a). Levels of the indicated proteins in the combined extracts of supernatants and cells were determined by western blotting. Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. f Primed MafB+/+ and MafB−/− PMs were treated as in (a). Triton X-100 soluble and insoluble extracts were prepared. Insoluble extracts were cross-linked and ASC oligomerization determined. g, h Primed MafB+/+ and MafB−/− PMs were stimulated with or without Nigericin for 1 h. Cells were then fixed with 4% PFA and subjected to immunofluorescence staining for ASC. Scale bars: 100 μm (g). ASC specks in the cells were quantified (h). n = 5. i–k Primed MafB+/+ and MafB−/− PMs (i, k) and BMDMs (j) were stimulated with ATP or Nigericin for 1 h. The levels of cleaved Caspase 1 (p20), pro-Caspase 1, and cleaved Gasdermin D (p30) in the supernatants and cells were determined by western blotting. Mean ± SD; **P < 0.01, ***P < 0.001. Representative of three independent experiments.