Fig. 4: IIC is a functional ligand for TARM1 and controls GM-DC maturation in a TARM1-dependent manner. | Nature Communications

Fig. 4: IIC is a functional ligand for TARM1 and controls GM-DC maturation in a TARM1-dependent manner.

From: TARM1 contributes to development of arthritis by activating dendritic cells through recognition of collagens

Fig. 4

a Bindings of TARM1-Fc to WT GM-DCs were analyzed by flow cytometry at day 8 after GM-CSF treatment. IgG Fc was used as a negative control. b Bindings of TARM1-Fc and IgG Fc to plate-coated IC, IIC, and BSA were analyzed by solid-phase binding assay. c Expression levels of Col1a1 and Col2a1 in GM-DCs, osteoblasts, and MEFs were analyzed by qPCR. d Cell surface expression of IC and IIC on GM-DCs at day 8 was analyzed by flow cytometry using specific antibodies. e Binding of TARM1-Fc to non- or collagenase-treated GM-DCs was analyzed by flow cytometry. IgG Fc was used as a negative control. f Whole GM-DC lysate was incubated with TARM1-Fc- and IgG Fc-beads, and after collection of beads, proteins were recovered. Then these proteins were immunoblotted with anti-IIC antibodies. Whole-cell lysate (WCL) was also immunoblotted with anti-IIC and anti-β-actin antibodies. g Expression of DC activation markers (CD86 and I-A/I-E) on WT and Tarm1–/– GM-DCs was analyzed by flow cytometry after stimulation with different concentration of soluble IIC for 24 h. h Cytokine concentrations (TNF, IL-6, and IL-10) of culture supernatants in g were determined by flow cytometry with cytometric beads. i GM-DCs at day 8 after GM-CSF treatment were stimulated with plate-bound IIC (1 μg/ml) for 24 h in the absence (PBS) or presence of TARM1-Fc, Fc, or TARM1-Flag (1, 3, and 10 μg/ml). The expression levels of I-A/I-E and CD86 in CD11c+ cells before and after IIC stimulation were analyzed by flow cytometry. The expression levels of these molecules on GM-DCs at day 8 before IIC treatment were shown as Day 8. All the data are representative of three independent experiments, except two experiments in i. Mean ± SD of triplicate wells. N.D. not detected. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed unpaired t test). Source data are provided as a Source data file.

Back to article page