Figure 5: IL-17RD disrupts signalling downstream of TIR adaptors.

(a) WT and Il17rd^−/− BMDMs were stimulated for 0, 5, 20 and 60 min with LPS (10 ng ml⁻¹) and cell lysates were subject to immunoprecipitation with an anti-MyD88 antibody and subjected to immunoblotting with indicated antibodies. (b,c) WT and Il17rd^−/− BMDMs or BMDCs were treated for (b) 0, 5, 30 and 60 min with LPS (10 ng ml⁻¹) or (c) 0, 30, 90 and 180 min poly(I:C) (5 μg ml⁻¹), respectively. Cell lysates were subject to SDS treatment, immunoprecipitation with an anti-TRAF6 antibody and probed by immunoblotting with indicated antibodies. (d,e,g) HEK293 cells were transfected for 24 h with MyD88-FLAG with (d) TRAF6-HA with IL-17RD-Myc, IL-17RD ΔC-Myc or IL-17RD SEFIR-Myc (total 3 μg) or (e) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR ΔBox1 or IL-17RD SEFIR ΔBox3 or (g) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR T496P, IL-17RD SEFIR K497R, IL-17RD SEFIR L504F or IL-17RD SEFIR T496P/K497R. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (f,h) Assay of NF-κB-regulated luciferase expression in HEK 293 cells transfected with MyD88 (50 ng) with (f) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR ΔBox1 or IL-17RD SEFIR ΔBox3 (100 ng) or (h) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR T496P, IL-17RD SEFIR K497R, IL-17RD SEFIR L504F or IL-17RD SEFIR T496P/K497R (100 ng). Data are (a–e,g) representative of three independent experiments or (f,h) are presented 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. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 10. NS, not significant.