Cbl-b negatively regulates in vitro CLR-mediated innate immune responses. (A) Nuclear p65 in WT and Cbl-b–deficient BMDMs that were stimulated with 40 µg/ml of precoated α-mannans for different times. The data shown are representative of three independent and reproducible experiments. (B) Quantification grayscale analysis of p65 versus internal control proliferating cell nuclear antigens (PCNA) in A. (C) ELISA results for TNF in supernatants of WT and Cbl-b–deficient BMDMs, which were stimulated with precoated α-mannans at the indicated concentrations for 12 h. (D) ELISA results for IL-6, IL-1β, and IL-12p40 in supernatants of WT and Cbl-b–deficient BMDMs, which were stimulated with 40 µg/ml of precoated α-mannans for 12 h. (E and F) ELISA results for TNF and IL-6 in supernatants of WT and Cbl-b–deficient BMDMs, which were stimulated with 40 µg/ml of precoated LAM (E) or TDM (F) at the indicated concentrations for 12 h. (G) ELISA results for TNF and IL-6 in supernatants of WT and Cbl-b–deficient BMDMs, which were pretreated with or without anti–Dectin-3 antibody (α–Dectin-3), α–Dectin-2, or an isotype-matched control IgG for 30 min before stimulation with C. albicans hyphae (MOI = 0.1) for 12 h. Usti., unstimulated. (H) ELISA results for TNF and IL-6 in supernatants of WT, Cbl-b KO, CARD9 KO, or Cbl-b/CARD9 dKO BMDMs, which were stimulated with C. albicans hyphae (MOI = 0.01 or 0.1) for 12 h. Data are means ± SD of triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test or ANOVA).