Fig. 1: Syntenin-1 binds to the C-terminal tail of Unc93b1 and restricts TLR7 signaling.

(a) Intracellular cytokine staining of TNFα in macrophage lines expressing the indicated Unc93b1 alleles and stimulated with CpG-B (100 nM), R848 (10 ng/ml), ssRNA40 (2.5 µg/ml), PolyIC (20 µg/ml), or LPS (10 ng/ml). Shaded histograms are unstimulated controls. HR: Unc93b1^H412R. (b) TNFα production, measured by ELISA, from the indicated RAW macrophage lines after stimulation for 8h with R848 (10 ng/ml), ssRNA40 (1 µg/ml), CpG-B (25 nM), or LPS (50 ng/ml). Data are mean ± s.d., n=2 biological replicates. PKP: Unc93b1^PKP/AAA. (c) Topology of Unc93b1 with the C-terminal regulatory region indicated in orange. (d) Immunoprecipitation (IP) of MyD88 from RAW macrophage lines expressing the indicated Unc93b1-Flag alleles and stimulated with R848 (500 ng/ml) followed by immunoblot for IRAK2. Input levels of Myd88 and IRAK2 in whole cell lysates (WCL) are also shown. (e) Silver stained SDS-PAGE gel of purified Unc93b1-Flag complexes from phagosomes of RAW macrophages expressing the indicated Unc93b1-Flag alleles. The 32kD protein corresponding to Syntenin-1 is indicated. (f) Purified Unc93b1-Flag complexes described in (e) were immunoblotted for Syntenin-1. (g) Syntenin-1 binding to Unc93b1 was measured by Flag IP followed by immunoblot for Syntenin-1 from the indicated RAW macrophage lines stimulated with R848 (0.5 μg/ml). (h) Interaction between Syntenin-1 and Unc93b1 was measured as described in (g) from Unc93b1^WT RAW macrophages stimulated with R848 (0.5 μg/ml) or CpG-B (0.5 μM). (i) NFκB activation in HEK293T cells transiently expressing TLR7 and increasing amounts of Syntenin-1 was measured using a dual luciferase reporter assay. Cells were stimulated with R848 (50 ng/ml) for 16h prior to harvest. RLUs: relative luciferase units, normalized to Renilla expression (n=3 biological replicates). One-way ANOVA, ***p<0.0001. All data are representative of at least three independent experiments.