Attenuated caspase-1 activation and IL-1β secretion in Dapk^−/− macrophages. (A) Normal expression of NLRP3, ASC, and procaspase-1 in DAPK-null macrophages. Bone marrow–derived macrophages (BMM) from normal littermate control (WT) and Dapk^−/− mice were analyzed for the protein expression of DAPK, NLRP3, ASC, procaspase-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B-C) Reduced IL-1β secretion in DAPK-null macrophages. Bone marrow–derived macrophages from WT and Dapk^−/− (−/−) were primed with LPS (0.5 μg/mL) (B), Pam3CSK4 (Pam3, 1 μg/mL), or R848 (2 μg/mL) (C) for 4 hours, followed by incubation with ATP (5mM) for 60 minutes (B-C), or nigericin (10μM), MSU (150 μg/mL), alum crystal (500 μg/mL) (B) for 6 hours. The secreted IL-1β in the supernatants was determined by ELISA. (D) Normal TNF-α secretion in DAPK-deficient macrophages. Bone marrow–derived macrophages from WT and Dapk^−/− mice were stimulated with LPS, Pam3CSK4, and R848 for 6 hours, and TNF-α secreted measured by ELISA. (E-G) DAPK deficiency decreased caspase-1 activation. Macrophage and supernatants from LPS-primed and MSU-stimulated (E), Pam3CSK4-primed and ATP stimulated (F), and R848-primed and ATP stimulated (G) macrophages were collected before and after priming, and after MSU or ATP treatment. Cells were lysed to generate total cell lysates (TCL). Supernatants (SUP) were precipitated with cold (−20°C) acetone, and dissolved in ristocetin-induced platelet aggregation buffer. Both supernatant precipitates and cell lysates were resolved by SDS-PAGE. The amounts of pro-IL-1β, mature IL-1β (p17), NLRP3, ASC, procaspase-1, active caspase-1 (p10), and GAPDH in SUP and TCL were determined by immunoblotting. (B-D) Error bars represent SD of a specific experiment with triplicate samples. All experiments have been repeated at least 3 times with similar results. *P < .05, **P < .01, ***P < .001 for paired t test.