Fig 1: Silencing of TIRAP gene inhibits nuclear translocation of IRF5 in 60 min after CL075 stimulation. Experiments were performed on human MDMs (n = 6 donors). (a) TIRAP-silencing efficacy was quantified with RT-qPCR from parallel wells of CL075 stimulated cells using non-stimulated cells for normalization (fold = 1.0). Control or TIRAP-silenced cells were stimulated with CL075 (2 μg/mL) for one hour, followed by fixation of cells, double staining of IRF5 (b) and NF-kB (p65/RelA) (c), DNA staining by Hoechst 3342 for nuclei visualization, and quantitative imaging by high-content screening (Olympus Scan^R system). The level of nuclear IRF5 (b) and p65 (c) was calculated as the percentage of positively stained nuclei multiplied by the mean fluorescence-intensity value (MFI) of the positively stained nuclei. In non-stimulated cells, the background-staining levels (%pos × MFI) for nuclear IRF5 and p65 were <15 and <73, respectively. (d,e) Representative immunofluorescent images of non-stimulated (NS) and CL075 stimulated cells used for quantification of IRF5 (red channel) and p65 (green channel) in nuclei (blue channel). Scale bar shown in overlay represents 50 µm. Statistical significance was examined with paired t-test (** p < 0.01, **** p < 0.001).
Fig 2: TIRAP silencing significantly inhibits TLR8-mediated IFNβ and IL12 p70 secretion by primary human macrophages. IFNβ and TNF secretion in 6–8 consecutive experiments with cells from different donors were analyzed by specific ELISA kits, with other cytokines’ secretion addressed by BioPlex assays. Statistical significance evaluated using Wilcoxon matched-pairs signed-rank test, presented as mean with SD, significance levels—* p < 0.05, ns—non-significant.
Fig 3: TIRAP silencing attenuates cytokine production from MDMs challenged with clinical isolates of E. coli (ECO), while affecting mainly IL-12A induction by Group B streptococcus (GBS) and pro-inflammatory cytokine induction by S. aureus (SAU). MDMs (5–6 donors in consecutive experiments) were pre-treated with TIRAP siRNA or control oligo and incubated with LPS (100 ng/mL), CL075 (1 µg/mL), or live bacteria (GBS 248, SAU 17-2, and ECO 18-1) for a total time of four hours. The doses of bacteria were 1 × 105/mL (e5) and 1 × 106/mL (e6). This roughly corresponds to MOI 0.01 and 0.1 for GBS, MOI 0.02 and 0.2 for SAU, and MOI 0.1 and 1.0 for ECO. Gene expression was determined by RT-qPCR, normalized to untreated sample, and presented as a mean relative fold change +SEM. Statistical testing was done with 2-way RM-ANOVA and post-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Fig 4: TIRAP is recruited to TLR8-initiated MyD88 and IRAK1/4-signaling complex. Endogenous TIRAP was immunoprecipitated for four hours from lysates (whole cell lysates—WCLs) of human MDMs: untreated or stimulated by LPS (100 ng/mL) or CL075 (2 µg/mL) for indicated time. LPS stimulation was applied as a positive control for TIRAP recruitment to the activated Myddosome. Cellular lysates were analyzed in parallel to control for input, with WB for MyD88, IRAK1, IRAK4, and TIRAP. A representative experiment is shown from a total of four consecutive experiments with different donors.
Fig 5: Silencing of TIRAP consistently inhibits TLR8-mediated phosphorylation of Akt S473. (a) Western blotting of lysates from MDMs treated with a control oligo or TIRAP-specific siRNA oligo and stimulated with 100 ng/mL LPS or 2 μg/mL CL075. The antibodies used are indicated on the figure, and GAPDH or PCNA are equal-loading controls. (b) Graphs show quantifications of protein levels relative to GAPDH or PCNA for CL075-stimulated cells and (c) LPS-stimulated cells. Representative image and graphs for one of four donors. Densitometry analysis and normalization to loading control was done using LiCor Odyssey software.
Supplier Page from Thermo Fisher Scientific for TIRAP Antibody