Figure 3: ASC is dispensable for IL-1β secretion by the NLRP1b inflammasome.
(a–c) B6, ASC−/−, B6Nlrp1b+ and B6Nlrp1b+ASC−/− BMDMs were primed with 5 μg ml−1 LPS for 3 h and subsequently stimulated with 20 μM nigericin for 60 min (a) or transfected with 1 μg dsDNA for 24 h (b) or infected with S. typhimurium (m.o.i. 10) for 3 h in the absence of LPS priming (c) before secreted IL-1β levels were determined in culture supernatants. (d–g) B6, B6Nlrp1b+, B6Nlrp1b+ASC+/− and B6Nlrp1b+ASC−/− BMDMs were left untreated or pretreated with 5 μg ml−1 LPS for 3 h prior to being exposed to different concentrations of LeTx (d,e, 10 μg PA+10 μg LF) or (f,g, 500 ng PA+250 ng LF) for another 3 h. Secreted IL-1β levels were determined in culture supernatants by ELISA and lysates were immunoblotted for IL-1β. (h,i) LPS-primed B6, B6Nlrp1b+ and B6Nlrp1b+ASC−/− BMDMs were pre-incubated with 50 μM ac-YVAD-cmk for 30 min prior to LeTx treatment for 3 h (h, 10 μg PA+10 μg LF; i, 500 ng PA+250 ng LF). Secreted IL-1β levels were determined in culture supernatants. (j,k) B6Nlrp1b+ and B6Nlrp1b+ Casp1−/− BMDMs were primed with 5 μg ml−1 LPS for 3 h followed by LeTx treatment (high, 10 μg PA+10 μg LF; low, 500 ng PA+250 ng LF) for another 3 h. Levels of secreted IL-1β (j) and IL-18 (k) were determined in culture supernatants. (l–n) BALB/C and 129SVEV57 BMDMs were primed with 5 μg ml−1 LPS for 3 h prior to LeTx treatment (10 μg PA+10 μg LF) for another 3 h. Lysates were immunoblotted for the indicated proteins (l) and secreted IL-1β and IL-18 were determined in culture supernatants by ELISA (m,n). Data are shown as mean±s.d. from a single representative experiment of three experiments, with each condition performed in triplicate.
