Fig. 7.

Exchange of the PYD with the CARD domain leads to constitutive activation. a Chimeric CARD-NLRP3 variants and b corresponding interacting domains (PYD, CARD) were expressed in Nlrp3^−/− iBMDMs by doxycycline (a: 0, 0.005, 0.01, 0.02, and 0.05 μg/ml; b: 0, 0.01, 0.05, 0.1, 0.5, 1, and 5 μg/ml) and primed with LPS (100 ng/ml) and then tested for their constitutive and nigericin-induced activation (5 μM, 1 h, note that 0 μg/ml dox is omitted in the latter case), as followed by IL-1β release. c For ASC speck labeling, cells were primed with doxycycline (0.5 μg/ml) and LPS (100 ng/ml) for 10 h after which the cells were fixed and labeled with an antibody against ASC. The formation of ASC specks in primed and nigericin-treated (1 h, 10 μM) cells harboring NLRP3 is shown as the control. The bar represents 10 μm. d CARD-NLRP3 chimeras were introduced into Asc^−/− iBMDMs, and expression of the chimeras was induced with doxycycline. Constitutive activation was followed by measuring IL-1β release. Representative of 3 (a, b) or 2 (c, d) independent experiments is shown. The mean and the s.e.m. of 2 or 3 (a, b, d) biological replicates are shown. n.d. – below detection limit