Figure 6: LPS induces in situ association of IL-17RD with MyD88.

(a) PBMCs from human donor blood were treated with LPS (100 ng ml⁻¹) for 0, 1, 5, 20 and 60 min and cell lysates were subject to immunoprecipitation with an anti-IL-17RD antibody. Cell lysate and immunoprecipitate samples were immunoblotted with indicated antibodies. (b) PBMCs from human donor blood were transfected with scrambled or IL-17RD-specific siRNA and cultured for 48 h prior to treatment in the absence or presence of LPS (100 ng ml⁻¹) for 20 min. Cells were fixed to slides and subjected to Duolink in situ proximity ligation assay using anti-IL-17RD and anti-MyD88 antibodies. IL-17RD/MyD88 interactions are visible as red fluorescence of the Duolink detection reagents with nuclear staining in blue (DAPI). Images were captured using the × 20 objective of a fluorescent microscope with scale bars representing 50 μm. The histogram represents the area of red Duolink signal as a percentage of area of blue DAPI signal from five random images from each of three independent experiments. Probe alone indicates areas of signals from cells subjected to ligation assay, but in the absence of anti-IL-17RD and anti-MyD88 antibodies (five random images from each of the two independent experiments). Data are presented as the mean ±s.e.m. and were subjected to a two-tailed paired Student’s t-test. *P<0.05; **P<0.01. The lowest-right panel demonstrates knockdown of IL-17RD expression by IL-17RD-specific siRNA as determined by immunoblotting of cell lysates from cells treated with LPS with or without prior transfection with IL-17RD-specific siRNA or a scrambled sequence version of this siRNA. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 11.