LUBAC is required for NLRP3 inflammasome activation in BMDMs. (a) IL-1β secretion as determined by ELISA of WT or HOIL-1L^−/− BMDMs primed with LPS overnight and stimulated with nigericin, silica, MSU, MDP followed by ATP, dsDNA transfected by lipofectamine, or infected with adenovirus-Flagellin as indicated. Unprimed BMDMs were infected with L. pneumophila at indicated multiplicities of infection (MOIs). Data are presented as the mean ± SEM by a Student’s t test (biological n = 3 and are representative of two independent experiments; *, P < 0.00033; #, P = 0.011). (b) Protein expression and caspase-1 cleavage of WT or HOIL-1L^−/− BMDMs untreated or primed with LPS and stimulated with nigericin was analyzed by immunoblotting. (c) BMDMs primed with LPS and stimulated with nigericin were treated with FAM-YVAD-FLICA fluorescent caspase-1 substrate during MDP treatment, fixed, and then stained for caspase-1. The percentage of FLICA-positive cells was determined for 250 total cells. Arrows point to FLICA-stained spots. Bars, 20 µm. (d–h) WT or HOIL-1L^−/− BMDMs were complemented with lentiviral vectors expressing mutants described in d and analyzed for: (e) IL-1β secretion by ELISA (biological n = 3 and analytical n = 2; data presented as the mean ± SEM; **, P < 0.0075; #, P < 0.013 by a Student’s t test); (f) mRNA quantification by qRT-PCR; (g) protein expression by immunoblot; and (h) quantification of FLICA staining for active caspase-1. All data are representative of three independent experiments except for c and h, which depict combined data for all three experiments. Protein size markers are indicated in kD.