Figure 6: ASC is dispensable for post-translational modification of active caspase-1.

(a,b) B6, B6^Nlrp1b+ and B6^Nlrp1b+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 (a, 10 μg PA+10 μg LF) or (b, 500 ng PA+250 ng LF) for another 3 h. Cell lysates were immunoblotted for caspase-1. (c,d) B6^Nlrp1b+ and B6^Nlrp1b+Casp1^−/− (c), or BALB/c and BALB/c Casp1^−/− BMDMs (d) were treated with 5 μg ml⁻¹ LPS for 3 h and subsequently exposed to 10 μg PA+10 μg LF (high) or 500 ng PA+250 ng LF (low) for another 3 h. Cell lysates were immunoblotted for caspase-1. (e–g) Lysates of B6 BMDMs were immunoblotted for caspase-1 after cells have been primed with 5 μg ml⁻¹ LPS for 3 h and subsequently stimulated with 5 mM ATP or 20 μM nigericin for 30 min (e), infected with F. tularensis (m.o.i. 30) for 3 h (f), or infected with S. typhimurium (m.o.i. 10, 5 and 1) for 3 h (g). (h,i) LPS-primed B6^Nlrp1b+ (h) and B6^Nlrp1b+ASC^−/− (i) BMDMs were pretreated with 50 μM Ac-YVAD-cmk for 30 min prior to being exposed to 10 μg PA+10 μg LF (High), or 500 ng PA+250 ng LF (Low) for another 3 h. Cell lysates were immunoblotted for caspase-1. (j,k) LPS-primed BALB/c BMDMs were exposed to 500 ng PA+250 ng LF ml⁻¹ for 120 or 190 min, respectively (j), or treated with 5 mM ATP for 30 min (k) before caspase-1 was immunoprecipitated using caspase-1 nanobody clone R2S40. Immunoprecipitates and lysates were immunoblotted for the indicated proteins. Data are representative of results from at least three independent experiments.