Distinct ASC isoforms can function as either activating or inhibitory inflammasome adaptor. (A) Inflammasomes were reconstituted in HEK293 cells by transient transfection of pro-IL-1β, pro-caspase 1, ASC, ASC-b, ASC-c, ASC-d, in the absence (black bars) or presence (gray bars) of the constitutive active NLRP3R260W, as indicated. Culture supernatants were analyzed for secreted IL-1β by ELISA 36 hours post transfection. (B) The ASC deficient RAW264.7 mouse macrophage cell line was stably transfected with empty vector, myc-tagged ASC, or myc-tagged ASC-b and analyzed for IL-1β release in resting cells (black bars) and following LPS (300 ng/ml)/ATP (5 mM) activation (gray bars). (C) Inflammasomes were reconstituted in HEK293 cells as shown in Figure 6A. Secreted IL-1β was analyzed by ELISA. All experiments were performed in triplicates (n = 3, +/- SD). (D) Control THP-1 cells (Ctrl) or THP-1 cells stably expressing high levels of ASC-c (#1) or low levels of ASC-c (#2) were treated with LPS (300 ng/ml) for 16 hours and analyzed for IL-1β release. Expression of ASC-c was determined by immunoblot. (E) Control J774A1 cells (Ctrl) or J774A1 cells stably expressing ASC-c were treated with LPS (300 ng/ml) for 16 hours, pulsed with 3 mM ATP for 15 minutes and analyzed for IL-1β release. Experiments in D and E were performed in triplicates (n = 2, +/- SD). Expression of ASC-c was determined by immunoblot. Note that the lysates from THP-1 and J774A1 cells were separated on the same gel and are the same exposure time.