Figure 4: SEFIR domain is critical for inhibitory effects of IL-17RD.

(a) HEK293 cells were transfected for 24 h with (a) MyD88-FLAG, Mal-FLAG and IL-17RD-Myc (1 μg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (b) HEK293 cells were transfected for 24 h with MyD88-GFP and IL-17RD-RFP and visualized for expression and localization using confocal microscopy. Cells were also incubated with nuclei-staining DAPI. Confocal images were captured using the × 63 objective (oil immersion) on the UV Zeiss 510 Meta System laser scanning microscope equipped with the appropriate filter sets and analysed using the LSM 5 browser imaging software (scale bar, 20 μm). (c) HEK293 cells were transfected for 24 h with MyD88-FLAG with IL-17RD-Myc or IL-17RD ΔC-Myc (1 μg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (d) Assay of NF-κB-regulated luciferase expression in HEK293 cells transfected with MyD88 (50 ng) with IL-17RD-Myc or IL-17RD ΔC-Myc (50 ng). (e–g) HEK293 cells were transfected for 24 h with (e) TLR4-FLAG and IL-17RD-Myc or IL-17RD ΔC-Myc, (f) MyD88-FLAG and IL-17RD-Myc, IL-17RD ΔC-Myc and IL-17RD SEFIR-Myc or (g) TLR4-FLAG and IL-17RD SEFIR-Myc (1 μg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (h) Assay of NF-κB regulated luciferase expression in HEK293 cells transfected with MyD88 (50 ng) and IL-17RD-Myc or IL-17RD SEFIR-Myc (100 ng). Data are (a–c,e–g) representative of three independent experiments or (d,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. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 9. NS, not significant.