Activation of inflammasome with LPS and R837 induces IL-1β secretion but not degranulation in MCs. (A) Semiquantitative RT-PCR analysis of Nlrp3, Il1b, Asc, Casp1, and Tlr4 in mouse BMCMCs stimulated with 100 ng/ml LPS. 5 µM MG132 was added 30 min before LPS stimulation. (B) Expression of specific mRNAs after stimulation for 3 h with 100 ng/ml LPS in BMCMCs was quantified by real-time PCR and normalized to Gapdh expression (×10⁴). (C) BMCMCs were pretreated with LPS for 15 h and stimulated by R837 for 30 min. IL-1β levels in supernatants were measured by ELISA. (D) Immunoblot analysis of cultured supernatant (SN) and cell lysate (CL) from BMCMCs incubated with the indicated stimuli. (E and F) BMCMCs were stimulated with 100 ng/ml LPS for 15 h, and the amounts of TNF-α and IL-6 in culture supernatants were measured by ELISA. (G) Degranulation of BMCMCs was assessed by the release of β-hexosaminidase. Error bars represent means ± SD of triplicates. All results are representative of at least three separate experiments. *, P < 0.005; and **, P < 0.001 compared with untreated BMCMCs. NS, not stimulated.