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. 2012 Jul 18;189(5):2131–2137. doi: 10.4049/jimmunol.1201349

FIGURE 6.

FIGURE 6.

Real-time monitoring of IL-1β processing in macrophages. (A and B) R-proIL1β-V BRET kinetic in macrophages. Macrophages were transfected and treated with LPS (1 μg/ml for 4 h). BRET was measured every 1 min in living macrophages after stimulation with 5 μM nigericin. BRET decrease was not observed in immortalized macrophages from ASC or NLRP3 knockout mice or in macrophages incubated with a caspase-1 inhibitor (Ac-YVAD-AOM, 100 μM), glyburide (150 μM), BAPTA-AM (100 μM), or an extracellular solution with high concentration of potassium (130 mM KCl). (C) BRET kinetic in BMDM. Immortalized BMDM from WT mice were transfected with R-proIL1β-V or R-proIL1β-D27A/D116A-V constructs and treated with LPS (1 μg/ml for 4 h). BRET was measured every 30 s before and after stimulation with 5 μM nigericin. (D) BRET kinetic in J774A.1 and BMDM macrophage cell lines. Macrophages were transfected with R-proIL1β-V and treated as described above. BRET was measured every 1 min. Results in (A–D) are mean ± SEM of n ≥ 3 experiments and n = 3 wells per experiment.