LRRK2 is critical for NLRC4 inflammasome activation. (a and b) LPS-primed WT and Lrrk2^−/− peritoneal macrophages were treated with 1 µg/ml LFn-PrgJ and anthrax-protective antigen (PA; a) or 1 µg/ml LFn-flagellin + anthrax-protective antigen for 1 h (b). Cell lysates and culture supernatants (Sup) were collected and immunoblotted with the indicated antibodies. (c) Peritoneal macrophages from littermate control (WT) and Lrrk2^−/− mice were infected with S. Typhimurium at an MOI of 100 for 2 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. (d) ELISA of TNF-α in cell-free supernatants from WT and Lrrk2^−/− peritoneal macrophages that were either infected with S. Typhimurium at an MOI of 100 for 2 h or pretreated with LPS (500 ng/ml) for 4 h followed by stimulation with LFn-flagellin + anthrax-protective antigen (1 µg/ml), LFn-PrgJ anthrax-protective antigen (1 µg/ml), ATP (5 mM), or nigericin (20 µM) for 1 h. (e) WT and Lrrk2^−/− peritoneal macrophages were primed with 500 ng/ml LPS for 4 h and then treated with ATP (5 mM) or nigericin (20 µM) for 1 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. “Ctrl” indicates the control group. (f) ELISA of IL-1β in cell-free supernatants from WT and Lrrk2^−/− peritoneal macrophages that were treated as stated in d. Data are shown as means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; determined by Student’s t test; all conditions were determined in triplicate. In each panel, data are representative of at least three independent experiments.