Fig. 6. Axl- and Mer-activating antibodies.

(a) BMDM cultures were pre-treated with 0.1 μM Dex or 10 μg/ml poly(I:C) for 18 h, and then stimulated with control antibody, α-Axl or α-Mer activating antibody for 20 min, with or without addition of 10 nM GAS-6 for the final 10 min. Receptor activation was assayed by immunoprecipitation and immunoblotting. Representative of two independent experiments.

(b) BMDM cultures were pretreated for 18 h with 0.1 μM Dex, 10 μg/ml poly(I:C) or 250 U/ml IFN-γ and then stimulated for 10 min with 10 nM of the indicated activating antibody. Representative of two independent experiments.

(c) Mice were injected IV with 10 μg of α-Axl antibody or control IgG, and spleens were collected at the indicated time points. Axl receptor phosphorylation was assayed by immunoprecipitation and immunoblotting. GAS-6 presence was assayed by immunoblotting of total spleen lysates. Representative of two independent experiments.

(d,e) Mice were injected IV with indicated dose of α-Axl or control IgG (d) or α-Mer or control IgG (e) for 1 h. Axl and Mer receptor phosphorylation was assayed by immunoprecipitation and immunoblotting. Representative of two independent experiments.

(f,g) BMDM cultures were pretreated for 24 h with 0.1 μM Dex (f) or 10 μg/ml poly(I:C) (g) and the phagocytosis assay was performed in the presence of 10 nM α-Mer (f) or α-Axl (g) activating antibody and indicated concentrations of GAS-6. Data is presented as mean ± s.d. from two independent experiments, each done for duplicate cultures for each condition.

(h,i) WT mice were injected IP with saline or 10 μg LPS together with 10 μg of either control IgG or α-Axl activating antibody. Ifnb (h) and Ifna4 (i) induction in spleen was measured by RT-qPCR relative to Cyclophilin A expression and presented as fold of change. Each mouse is plotted as a separate data point. Mean is indicated. n=5 (saline IgG), n=4 (saline α-Axl), n=10 (LPS IgG), n=7 (LPS α-Axl),