NLRP3 inflammasome activation by EMCV viroporin 2B.

<p>(A) LPS-primed BMMs were infected with the lentivirus expressing EMCV 2A, 2B, or 2C, or GFP. Supernatants were collected at 24 h post infection and analyzed for IL-1β by ELISA. (B) HEK293T cells were transfected with the expression plasmid encoding Flag- or HA-tagged EMCV 2A, 2B, or 2C, or NLRP3 or empty vector. Samples were analyzed by immunoblot with rabbit polyclonal antibody against Flag or mouse monoclonal antibody against HA. (C) HeLa cells were transfected with the expression plasmid encoding Flag-tagged EMCV 2A, 2B, or 2C (green) and that encoding either pDsRed-monomer-Golgi or pDsRed2-ER (red), and observed with a confocal microscope at 24 h after transfection. (D) LPS-primed BMMs were stimulated with EMCV or nigercin for 24 h in the presence or absence of yVAD-CHO. Cells were stained with anti-NLRP3 (green) and anti-GM130 (Golgi marker) or anti-calnexin (ER marker) (red) and analyzed by a confocal microscopy. Mock-treated cells were also examined. Nuclei were visualized by staining with DAPI. (E) HeLa cells were transfected with the expression plasmid encoding Flag-tagged NLRP3 (green) and that encoding HA-tagged EMCV 2A, 2B, or 2C, or influenza virus M2 protein (red). Nuclei were visualized by staining with DAPI. Scale bars represent 10 µm. Data are representative of at least three independent experiments, and indicate the mean ± S.D (A). ***, <i>P</i><0.001.