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. 2011 Jul 29;12(9):971–979. doi: 10.1038/embor.2011.143

Figure 1.

Figure 1

Cytokine secretion patterns of c-Cbl-knockout dendritic cells. (A) Wild-type and Cbl-KO BMDCs were stimulated with the indicated TLR agonists (Ctrl, dimethylsulphoxide; TLR1/2, Pam3CSK4; TLR2, LTA; TLR3, poly(I:C); TLR4, LPS; TLR7, R837; TLR7/8, CL075; TLR9, CpG) overnight and the supernatants were collected for IL-12p70 measurement by ELISA. (B) Supernatants of cultured BMDCs with indicated genotypes were collected after 24 h of LPS stimulation for quantification of the indicated cytokines and chemokines by LINCOplex assay. Data were pooled from 3–4 pairs of WT and Cbl-KO samples (data represent average±s.e.). (C) WT and Cbl-KO BMDCs were stimulated with the indicated TLR agonists overnight and subjected to intracellular staining of IL-12p40. Cells were treated with brefeldin A and analysed on a BD LSRII cytometer. (D) WT and c-Cbl-deficient BMDCs were stimulated with LPS (1 μg/ml) at the indicated time points before preparation of total RNA. The relative amounts of IL-12p35, p40 and IL-10 were quantified by quantitative real-time polymerase chain reaction with predesigned primers. (E) WT BMDCs nucleofected with the indicated siRNAs (left panel) or paired WT and Cbl-KO BMDCs (right panel) were cultured overnight. Supernatants were collected for analysis of IL-12p70 production by ELISA. All data (except in B) are representative of at least three experiments with consistent results. Cbl-KO, c-Cbl-knockout; ELISA, enzyme-linked immunosorbent assay; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; IFN, interferon; KD, knockdown; LPS, lipopolysaccharide; ND, not determined; NS, not significant; siRNA, small-interfering RNA; TLR, Toll-like receptor; WT, wild type.