Figure 1: IL-17RD negatively regulates TLR signalling pathways.

(a,b) Assay of NF-κB-regulated luciferase reporter activity in HEK293 cells transfected with Myc-tagged IL-17RD (0–100 ng) and (a) MyD88, Mal, TRIF or TRAM (50 ng) with a NF-κB luciferase reporter plasmid (60 ng) or (b) with TRIF (50 ng) and PFR-luciferase (60 ng) with IRF3-Gal4 (30 ng) or IRF7-Gal4 (25 ng) constructs. TK Renilla was measured to determine transfection efficiency. (c,d) Supernatants were measured for (c) IL-8 or (d) RANTES production by sandwich ELISA. (e,f) ELISA of (e) TNF-α or (f) IL-8 from U373 or THP-1 cells, respectively, previously transduced with lentiviral-encoded control or IL-17RD-specific shRNA and treated with LPS (100 ng ml⁻¹) for 24 h. (g) Supernatants from U373 cells stably transduced with control or IL-17RD-specific shRNA measured for RANTES and IP-10 levels by ELISA in response to poly(I:C) (25 μg ml⁻¹). (h,i) Cell lysates from (h) THP-1 cells treated with LPS (100 ng ml⁻¹) for 0, 5, 15, 30, 60 and 240 min and (i) U373 cells treated with poly(I:C) (25 μg ml⁻¹) for 0, 45, 90, 120, 240 and 360 min, both stably expressing control and IL-17RD-specific shRNA, were subjected to immunoblotting with indicated antibodies. (h,i: inset panels) Cell lysates from THP-1 and U373 cells expressing control (Ctrl) or IL-17RD-specific shRNA (shRNA) were analysed for IL-17RD and β-actin expression with specific antibodies by immunoblotting. Overexpressed IL-17RD (oe) was used as a control from HEK293 and U373 cell lysates, respectively. Data are presented (a–g) as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test, *P<0.05; **P<0.01 or (h,i) are representative of three independent experiments. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 7.